Fine Structure of Spirochaeta stenostrepta, a Free-living, Anaerobic Spirochete

The fine structure of Spirochaeta stenostrepta strain Z1, a free-living anaerobic spirochete, was studied by electron microscopy. The organism possessed a coiled protoplasmic cylinder, an axial filament inserted subterminally, and a loosely fitting sheath which enclosed both the protoplasmic cylinder and the axial filament. The axial filament consisted of two fibrils partially overlapping in a 1-2-1 arrangement. The axial fibrils appeared to possess a sheath surrounding an inner core. Both inner core and sheath were apparently enclosed in a cross-striated tubular structure, which was itself surrounded by an outer sheath. The axial filament exhibited a basal hook. A disc- or mushroom-shaped structure, possibly consisting in part of cytoplasmic membrane, was observed at the insertion end of isolated filaments. The protoplasmic cylinder had a distinctive surface structure consisting of an array of tightly packed, longitudinally arranged helices measuring 2.0 to 2.5 nm in diameter. This layer of helices lay below the outer cell sheath and the axial filament. Ballistic disintegration loosened the helical array, causing individual helices or segments of helices to become separated from the cell. The function of this layer of helices is still obscure.     

Treponema bryantii sp. nov., a rumen spirochete that interacts with cellulolytic bacteria

A saccharolytic spirochete that associated and interacted with cellulolytic bacteria was isolated from bovine rumen fluid. Isolation was accomplished by means of a procedure involving serial dilution of a sample of rumen fluid into a cellulose-containing agar medium. Clear zones appeared within the medium as a result of cellulose hydrolysis by rumen bacteria. The saccharolytic spirochete and a cellulolytic bacterium later identified as a strain of Bacteroides succinogenes were isolated from the clear zones. The spirochete did not utilize cellulose, but grew in coculture with the cellulolytic bacterium in cellulose-containing media. When cocultured in these media the spirochete used, as fermentable substrates, soluble sugars released from cellulose by the cellulolytic bacterium. In cellulosecontaining agar medium the spirochete enhanced cellulose breakdown by the B. succinogenes strain. Electron microscopy showed that the helical spirochete cells possessed an outer sheath, a protoplasmic cylinder, and two periplasmic fibrils. Under a CO₂ atmosphere, in a reduced medium containing inorganic salts, rumen fluid, glucose, and NaHCO₃, the spirochete grew to a final density of 1.9×10⁹ cells/ml. Succinate, acetate, and formate were products of the fermentation of glucose by growing cells. CO₂ (HCO₃ ^-), branched short-chain fatty acids, folic acid, biotin, niacinamide, thiamine, pyridoxal, and a carbohydrate were required for growth of the spirochete. The results of this study indicated that the rumen spirochete represents a new species of Treponema. It is proposed that the new species be named Treponema bryantii.Abbreviations cpm counts per minute - GC guanine plus cytosine - T_m melting temperature - PC protoplasmic cylinder - PF pertplasmic fibrils (axial fibrils) - OS outer sheath - ID insertion disk     

Electron Microscopy of the Spherical Body of Oral Spirochetes In Vitro: Further Studies

The surface and inner structure of the spherical bodies (SB) produced by the human oral treponeme strain G7201, similar to Treponema macrodentium, were studied by electron microscopy. Ultrathin sectioning and scanning techniques demonstrated that in the presence of a high concentration of sucrose, the outer envelope of one or both terminal ends of this oral spirochete changed into a swollen structure, the SB. Spirochetal cells adhered firmly to the surface of the resultant body. The membrane of the SB, i.e. the outer envelope, enclosed the coiled protoplasmic cylinder and five axial fibrils which were located between the envelope and the cylinder. Large expanded protoplasmic cylinders were observed, surrounded by a partially disrupted double membrane in some SBs. A number of frizzly fibrous structures, which differed from axial fibrils in number and shape, were also observed within these SBs. Except for abnormal or partially broken cylinders, the protoplasmic cylinders tended to be located close to the inner surface of the SB membrane, resulting in a central vacant space with occasional axial fibrils. These findings suggest that the oral spirochete produces an SB by terminal expansion of the outer envelope in the presence of high concentrations of sucrose. The outer envelope of the SB, which consists of two electron-dense layers, has the property of binding spirochetal cells to its outer layer and the protoplasmic cylinder and axial fibrils to the inner layer. Some protoplasmic cylinders were also observed to be swollen in the presence of high sucrose concentrations.     

Variable cellular composition of Chromatium in growing cultures

Summary A strictly anaerobic spirochete was isolated from a sample of marine mud. The organism possessed two axial fibrils entwined with the regularly coiled protoplasmic cylinder. An outer envelope or sheath enclosed both protoplasmic cylinder and axial fibrils. The spirochete grew in chemically defined media containing glucose, amino acids or NH₄Cl, sulfide, NaCl, vitamins, coenzyme A, and in-organic salts. A reducing agent, such as sodium sulfide or l-cysteine, as well as exogenous supplements of biotin, niacin and coenzyme A were required for growth. Pantothenate replaced coenzyme A as an exogenous growth factor, but the resulting cell yields were low. The spirochete grew in media prepared with sea water, but not in fresh water media containing less than 0.05 M NaCl (optimum concentration 0.35 M). Both Na⁺ and Cl^- were required. Carbohydrates served as fermentable substrates. Amino acids, sugar alcohols, tricarboxylic acid cycle intermediates, and other organic acids and alcohols were not fermented. Glucose was fermented to ethyl alcohol, acetate, CO₂, H₂, and small amounts of lactate, formate and pyruvate. The guanine + cytosine content of the DNA of the spirochete was 50.5 moles-% (buoyant density). It is proposed that the marine isolate be considered a new species and that it be named Spirochaeta litoralis.     

Treponema succinifaciens sp. nov., an anaerobic spirochete from the swine intestine

The morphology, the general physiological characteristics, and the energy-yielding metabolism of an obligately anaerobic spirochete isolated from the colon of a swine were studied. Electron microscopy showed that the helical spirochetal cells possessed an outer sheath, a protoplasmic cylinder, and 4 periplasmic fibrils in a 2-4-2 arrangement. The spirochete grew in an atmosphere of N₂ in prereduced media containing a carbohydrate, NaHCO₃, rumen fluid, yeast extract, peptone, l-cysteine, and inorganic salts. The spirochete fermented carbohydrates and required substrate amounts of CO₂ (HCO ₃ ^- ) for growth. Amino acids were not fermented. Major fermentation products of cells growing with glucose as the substrate and in the presence of CO₂ were acetate, formate, succinate, and lactate. Small amounts of 2,3-butanediol, pyruvate, and acetoin were also formed. Determinations of enzymatic activities in cell extracts, and of radioactivity in products formed by growing cells from [1-¹⁴C]glucose, indicated that this sugar was dissimilated to pyruvate via the Embden-Meyerhof pathway. The spirochetes used a coliform-type clastic reaction to metabolize pyruvate. Determinations of radioactivity in products formed from [¹⁴C]NaHCO₃ indicated that CO₂ was assimilated and used in succinate production. The guanine+cytosine content of the DNA was 36 mol%. This study indicates that this intestinal spirochete represents a new species of Treponema. It is proposed that the new species be named Treponema succinifaciens.Abbreviations cpm counts per minute - DTT dithiothreitol - EM Embden-Meyerhof - GC guanine plus cytosine - IgG immunoglobulin G - PC protoplasmic cylinder - PF periplasmic fibrils (axial fibrils) - OS outer sheath     

Anaerobic Spirochete from a Deep-Sea Hydrothermal Vent

An obligately anaerobic spirochete, designated strain GS-2, was selectively isolated from samples collected at a deep-sea (2,550 m) hydrothermal vent of the Galapagos Rift ocean floor spreading center. The morphological and physiological characteristics of strain GS-2 resembled those of Spirochaeta strains. However, strain GS-2 failed to grow consistently in any liquid medium tested. In addition, strain GS-2 grew more slowly and to lower yields than other Spirochaeta species. The occurrence of obligately anaerobic bacteria in hydrothermal vents indicates that the water in at least some of the vent areas is anoxic. The presence of strain GS-2 shows that these areas are favorable for anaerobic marine spirochetes.     

Spirochaeta caldaria sp. nov., a thermophilic bacterium that enhances cellulose degradation by Clostridium thermocellum

Two strains of obligately anaerobic, thermophilic spirochetes were isolated from cyanobacterial mat samples collected at freshwater hot springs in Oregon and Utah, USA. The isolates grew optimally between 48° and 52°C, and did not grow at 25° or 60°C. Both strains fermented various pentoses, hexoses, and disaccharides. Amino acids or cellulose did not serve as fermentable substrates for growth. H₂, CO₂, acetate, and lactate were end products of d-glucose fermentation. On the basis of physiological characteristics, guanine + cytosine content of DNA, and comparisons of 16S ribosomal RNA sequences, it was concluded that the two isolates were representatives of a novel species of Spirochaeta for which the name Spirochaeta caldaria is proposed. One of the two strains was grown in coculture with a thermophilic cellulolytic bacterium (Clostridium thermocellum) in a medium containing cellulose as the only fermentable substrate. In the coculture cellulose was broken down at a faster rate than in the clostridial monoculture. The results are consistent with the suggestion that interactions between cellulolytic bacteria and non-cellulolytic spirochetes enhance cellulose breakdown in natural environments in which cellulose-containing plant material is biodegraded.     

Isolation of Clostridium propionicum strain 19acry3 and further characteristics of the species

Two mixed cultures able to ferment acrylate to equimolar acetate and propionate were enriched from anaerobic sediments. From one of these mixed cultures a pure culture of a Gram-positive, obligately anaerobic bacterium was isolated. This strain, designated 19acry3 (= DSM 6251) was identified as belonging to the species Clostridium propionicum. Only a narrow range of organic compounds supported growth, including acrylate and lactate. Acrylate and lactate were fermented to acetate and propionate in a 1:2 molar ratio. When co-cultured with the non-acrylate-fermenting Campylobacter sp. strain 19gly1 (DSM 6222), the fermentation balance shifted to almost equimolar acetate and propionate. Strain 19acry3 was compared with Clostridium propionicum type strain X2 (DSM 1682). The two strains displayed similar phenotypic properties. The mol% G+C of DNA isolated from both strains was 36–37 (by thermal denaturation). Both strains displayed a characteristic fluorescence when observed by fluorescence microscopy. Cell-free extracts of both strains were examined by spectrophotofluorimetry. In both strains, two excitation peaks were observed at 378 and 470 nm. Excitation at either of these wavelengths resulted in an emission maximum at 511 nm.     

Isolation and characterization of a thermophilic acetotrophic strain of Methanothrix

An enrichment culture which converted acetate to methane at 60°C was obtained from a thermophilic anaerobic bioreactor. The predominant morphotype in the enrichment was a sheathed gas-vacuolated rod with marked resemblence to the mesophile Methanothrix soehngenii. This organism was isolated using vancomycin treatments and serial dilutions and was named Methanothrix sp. strain CALS-1. Strain CALS-1 grew as filaments typically 2–5 cells long, and cultures showed opalescent turbidity rather than macroscopic clumps. The cells were enclosed in a striated subunit-type sheath and there were distinct cross-walls between the cells, similar to M. soehngenii. The gas vesicles in cells were typically 70 nm in diameter and up to 0.5 m long, and were collapsed by pressures over 3 atm (ca. 300 kPa). Stationary-phase cells tended to have a higher vesicle content than did growing cells, and occasionally bands of cells were seen floating at the top of the liquid in stationary-phase cultures. Acetate was the only substrate of those tested which was used for methanogenesis by strain CALS-1, and acetate was decarboxylated by the aceticlastic reaction. The optimum temperature for growth of strain CALS-1 was near 60°C (doubling time=24–26 h), with no growth occurring at 70°C and 37°C. The optimum pH value for growth was near 6.5 in bicarbonate/CO₂ buffered medium and no growth occurred at pH 5.5 or pH 8.4. No growth was obtained below pH 7 when the medium was buffered with 20 mM phosphate. Strain CALS-1 grew in a chemically defined medium and required biotin. Sulfide concentrations over 1 mM were inhibitory to the culture, and growth was more rapid with 1 mM 2-mercaptoethane sulfonate (coenzyme M) or 1 mM titanium citrate as an accessory reductant than with 1 mM cysteine. It is likely that strain CALS-1 represents a new species in the genus Methanothrix.     

Clostridium grantii sp. nov., a new obligately anaerobic,alginolytic bacterium isolated from mullet gut

A gram-positive, motile, rod-shaped, strictly anaerobic, sporulating bacterium was isolated from an enrichment initiated with mullet gut contents. The organism grew optimally at 30°C and pH6.5, and at a salinity of 1–10³. Out of a variety of polysaccharides tested as growth substrates, only alginate supported growth in either semidefined or complex culture medium. The organism also grew on a variety of mono- and disaccharides. Moles product per 100mol of alginate monomer degraded were: acetate, 186; ethanol, 19; formate, 54; and CO₂, 0.19. Moles product per 100mol of hexose in cellobiose or glucose degraded were: acetate, 135; ethanol,61; formate, 63: and CO₂, 61. Hydrogen was not detectable during the incubations (detection limit, <10^-5atm) and propionate, butyrate, lactate, or succinate were not produced as fermentation end products (<2 mol per 100 mol of monomer). The G+C content of DNA from the bacterium was 30.2±0.3 mol%, and the cell walls contained the peptidoglycan component meso-diaminopimelic acid. A phylogenetic analysis of the 16S rDNA sequence indicated that the organism grouped closely with members of the RNA-DNA homology group 1 of the genus Clostridium. However, it differed from other species of the genus with regard to morphology, growth temperature optimum, substrate range, and fermentation pattern and is therefore designated as a new species of Clostridium; the type strain is A-1 (DSM 8605).     

Spirochaeta bajacaliforniensis sp. n. from a microbial mat community at Laguna Figueroa,Baja California Norte,Mexico

A new anaerobic spirochete was isolated from anaerobic muds beneath the laminated sediment in the evaporite flat at Laguna Figueroa, Baja California Norte, Mexico. The organism is a member of the stratified microbial community involved in the deposition of the laminated sediments in the lagoon. The size of the spirochete is 0.3 by 30 m, with a wave amplitude of 0.5 m and a wavelength of 1.25 m. The periplasmic flagella have a 1-2-1 arrangement. The outer membrane of the modified Gramnegative cell wall (the sheath) is irregularly crenulated and has a sillon. The growth medium contained yeast extract, trypticase, cellobiose, sodium thioglycolate and at least 20% natural seawater. Chemically defined artificial seawater media did not support growth. Optimal growth occurred with a seawater concentration of 80% at 36° C and a pH of 7.5. Glucose was fermented to acetate, ethanol, carbon dioxide and hydrogen. The guanine+cytosine content of the DNA was 50 mol %. the spirochete body reacts positively to antibodies raised against eukaryotic brain tubulin protein. On the basis of its free-living anaerobic habitat, its unique morphological and physiological characteristics and G+C ratio, it is proposed that this isolate be considered a new species and named Spirochaeta bajacaliforniensis.     

Isolation and characterization of an obligately anaerobic,pectinolytic, member of the genus Eubacterium from mullet gut

A gram positive, motile rod-shaped strictly anaerobic non sporulating bacterium was isolated from an enrichment initiated with mullet gut contents. The organism grew optimally at 30°C at pH 6.5 and at a salinity of 10/10³. Out of a variety of mono-, di-, and polysaccharides tested only pectin, cellobiose and starch actively supported growth in either semi defined medium or peptone-yeast extract (PY) medium. Galacturonic acid and maltose were less effective as substrates. Mol product per 100 mol of pectin monomer degraded were: acetate, 163; ethanol, 30; methanol, 88 and formate, 48. Per 100 mol of hexose in cellobiose or starch degraded, the amounts were acetate, 39; ethanol, 128 and formate, 41. Hydrogen was not detectable in the incubations (detection limit, <10^-5 atm) and propionate, butyrate, lactate or succinate were not produced as fermentation end-products (<2 mol per 100 mol monomer). The guanine plus cytosine content of DNA from the bacterium was 31 mol%, and the cell walls contained meso-diaminopimelic acid. A phylogenetic analysis of the organism by 16S rDNA sequencing and DNA-DNA homology indicated that the organism grouped more closely with several species of Clostridium than with Eubacterium. The phenotypic characteristics of the organism indicated that it did not fit within the genus Clostridium and more closely resembled Eubacterium. The organism is therefore designated as a species of Eubacterium; the type strain is P-1 (DSM 6788).     

Bacillus flavothermus,a newly isolated facultative thermophile

A sample from a hot spring on the northern island of New Zealand contained five different thermophilic bacterial strains. One strain with peculiar properties, i.e. the formation of dark yellow colonies at 30°C as well as at 70°C, was further characterized. It was found to be a gram-positive, facultatively aerobic, motile Bacillus species, with terminal endospores. According to the physiological properties the strain closely resembled B. coagulans. However, two typical characteristics were contradictory to this conclusion, namely the intense yellow pigmentation of the colonies and the range of growth temperature. The latter was found to reach from 30 to 70°C, with an optimum at 60°C under aerobic and at 65°C under anaerobic conditions. Growth at moderate temperatures was slower than at 60°C, but the final cell yields were almost equal. The strain can therefore be considered as facultatively thermophilic. The pigment, which was found to be located in the cytoplasmic membrane, was spectroscopically identified as a carotenoid.Because the characteristics of this strain did not correspond with any of the Bacillus species described thus far, we concluded, that we had isolated a novel strain, for which the name Bacillus flavothermus is proposed.     

Direct Measurement of Helical Cell Motion of the Spirochete Leptospira

Leptospira are spirochete bacteria distinguished by a short-pitch coiled body and intracellular flagella. Leptospira cells swim in liquid with an asymmetric morphology of the cell body; the anterior end has a long-pitch spiral shape (S-end) and the posterior end is hook-shaped (H-end). Although the S-end and the coiled cell body called the protoplasmic cylinder are thought to be responsible for propulsion together, most observations on the motion mechanism have remained qualitative. In this study, we analyzed the swimming speed and rotation rate of the S-end, protoplasmic cylinder, and H-end of individual Leptospira cells by one-sided dark-field microscopy. At various viscosities of media containing different concentrations of Ficoll, the rotation rate of the S-end and protoplasmic cylinder showed a clear correlation with the swimming speed, suggesting that these two helical parts play a central role in the motion of Leptospira. In contrast, the H-end rotation rate was unstable and showed much less correlation with the swimming speed. Forces produced by the rotation of the S-end and protoplasmic cylinder showed that these two helical parts contribute to propulsion at nearly equal magnitude. Torque generated by each part, also obtained from experimental motion parameters, indicated that the flagellar motor can generate torque >4000 pN nm, twice as large as that of Escherichia coli. Furthermore, the S-end torque was found to show a markedly larger fluctuation than the protoplasmic cylinder torque, suggesting that the unstable H-end rotation might be mechanically related to changes in the S-end rotation rate for torque balance of the entire cell. Variations in torque at the anterior and posterior ends of the Leptospira cell body could be transmitted from one end to the other through the cell body to coordinate the morphological transformations of the two ends for a rapid change in the swimming direction.     

Direct Measurement of Helical Cell Motion of the Spirochete Leptospira

Leptospira are spirochete bacteria distinguished by a short-pitch coiled body and intracellular flagella. Leptospira cells swim in liquid with an asymmetric morphology of the cell body; the anterior end has a long-pitch spiral shape (S-end) and the posterior end is hook-shaped (H-end). Although the S-end and the coiled cell body called the protoplasmic cylinder are thought to be responsible for propulsion together, most observations on the motion mechanism have remained qualitative. In this study, we analyzed the swimming speed and rotation rate of the S-end, protoplasmic cylinder, and H-end of individual Leptospira cells by one-sided dark-field microscopy. At various viscosities of media containing different concentrations of Ficoll, the rotation rate of the S-end and protoplasmic cylinder showed a clear correlation with the swimming speed, suggesting that these two helical parts play a central role in the motion of Leptospira. In contrast, the H-end rotation rate was unstable and showed much less correlation with the swimming speed. Forces produced by the rotation of the S-end and protoplasmic cylinder showed that these two helical parts contribute to propulsion at nearly equal magnitude. Torque generated by each part, also obtained from experimental motion parameters, indicated that the flagellar motor can generate torque >4000 pN nm, twice as large as that of Escherichia coli. Furthermore, the S-end torque was found to show a markedly larger fluctuation than the protoplasmic cylinder torque, suggesting that the unstable H-end rotation might be mechanically related to changes in the S-end rotation rate for torque balance of the entire cell. Variations in torque at the anterior and posterior ends of the Leptospira cell body could be transmitted from one end to the other through the cell body to coordinate the morphological transformations of the two ends for a rapid change in the swimming direction.     

The classification of free-living spirochetes

Summary The natural relationships among free-living and host-associated spirochetes were studied by investigating aspects of their morphology, physiology and DNA base composition. These studies indicated that the strictly and facultatively anaerobic, free-living spirochetes share characteristics which distinguish them from their host-associated counterparts and from the free-living leptospires.It is suggested that the strictly and facultatively anaerobic, free-living spirochetes be grouped in the genus Spirochaeta and that this genus include the following four species: S. plicatilis (type species), S. stenostrepta, S. zwelzerae (Treponema zuelzerae), and S. aurantia. The characteristics of these species are described.     

Thermoanaerobium acetigenum spec. nov., a new anaerobic,extremely thermophilic,xylanolytic non-spore-forming bacterium isolated from an Icelandic hot spring

An anaerobic, extremely thermophilic, xylanolytic nonspore-forming bacterium, strain X6B, was isolated from a 70°C Icelandic hot spring sediment. The bacterium was rod-shaped, 3.6–5.9 m long and 0.7 to 1.0 m wide, and cells grew singly, in pairs, and occasionally formed chains. The bacterium was nonmotile with no flagella. Cells from mid-to late exponential gowth-phase cultures stained gram-negative but had a gram-positive like cell wall structure in transmission electron photomicrographs. The bacterium grew between 50°C and 78°C with an optimum temperature at about 65°C to 68°C. Growth occurred between pH 5.2 and 8.5 with an optimum pH close to 7. During growth on beech wood xylan, glucose and d-xylose, the isolate produced CO₂, acetate and H₂ as major fermentation products, and a small amounts of ethanol; lactate was not produced. X6B did not reduce acetone to isopropanol or sulphate or thiosulfate to sulfide. The base composition of X6B's cellular DNA was 35.7 mol% guanine + cytosine. The properties of this strain do not fit any previously described species. The name proposed for the isolated bacterium was Thermoanaerobium acetigenum, spec. nov.     

Labidocera aestiva and L. scotti in Tamiahua Lagoon,Veracruz, Mexico

Labidocera aestiva and L. scotti were found in the Tamiahua Lagoon, Veracruz, Mexico. Fleminger (1957) found that the populations of these species may overlap geographically, although L. aestiva has affinity to the Carolinian province and L. scotti to the Caribbean province. This study describes the seasonal behavior and succession of this species in the Tamiahua Lagoon, a brackish water system with a high marine influence. A qualitative and quantitative analysis of samples was made in March, July, and September 1985 and January 1986. L. aestiva was found in temperatures below 26 °C in a wide salinity range. At temperatures above 26 °C and up to 32 °C, L. aestiva was present also with euryhaline character. In the Tamiahua Lagoon these two species did not overlap during this study. Both species are considered temporary inhabitants of this estuarine system in the Western Gulf of Mexico.     

Abundance and growth response of microalgae at Megalon Embolon solar saltworks in northern Greece: An aquaculture prospect

There is continuous interest in many countries in maintaining and manipulating the rich ecological value of hypersaline ecosystems for aquaculture. The Megalon Embolon solar saltworks (northern Greece) were studied in sites of increasing salinity of 60–144 ppt to evaluate Dunaliella salina abundance and microalgal composition, in relation to physical and chemical parameters. Cluster and ordination analyses were performed based on the biotic and abiotic data matrices. Using fresh aliquots from 60 and 140 ppt salinity waters, phytoplankton performance was appraised with flask cultures in the laboratory by varying the inorganic PO₄-P concentration at 23 °C and 30 °C. At the saltworks, among the most abundant microalgae identified were species of the genera Dunaliella, Chlamydomonas, Amphora, Navicula, and Nitzschia. Dunaliella salina populations were predominant comprising 5–22% of the total microalgal assemblages during spring, but only 0.3–1.0% during the summer, when grazing by Artemia parthenogenetica and Fabrea salina was intense. D. salina cell density in April–July was in the range of 0.4–12.5 × 10⁶ L⁻¹ with typical densities of 1.5–4.5 × 10⁶ L⁻¹. Overall, microalgal densities were high in salinities of ≥100 ppt when inorganic-P concentrations were ≥0.20 mg L⁻¹ within saltworks waters. Multivariate analysis of species abundance showed that algal growth responses were primarily related to variation in salinity and inorganic-P concentrations, but also to NO₃-N concentration. In the laboratory, experiments indicated effective fertilization and denser microalgal growth under high inorganic PO₄-P applications (4.0 and 8.0 mg L⁻¹) at 60 ppt salinity and 23 °C. The lower PO₄-P applications (0.6–2.0 mg L⁻¹) were more effective at 60 ppt salinity and 30 °C. At 140 ppt salinity, microalgal growth response was less obvious at any of the corresponding phosphorus concentrations or temperatures. In both salinity experiments, Dunaliella salina bloomed easily and was predominant among the microalgae. Our observations indicate that Dunaliella salina populations and the overall rich microalgal profile of the saltworks, along with their performance in laboratory mono–and mixed cultures hold promise for mass cultivation within the M. Embolon saltworks basins.     

Methanogenic Sarcina from an Anaerobic Microbial Community Degrading p-Toluene Sulfonate

The methanogenic strain MM isolated from an anaerobic microbial community degrading p-toluene sulfonate showed optimal values of temperature and pH for growth equal to 37°C and 6.3–6.9, respectively. The doubling times of the isolate grown on methanol, acetate, and methylamines under the optimal conditions were 8.8, 19.1, and 10.3–28.1 h, respectively. The growth of strain MM was observed only when the cultivation medium contained casamino acids or p-toluene sulfonate. The G+C content of the DNA of the isolate was 40.3 mol %. This, together with DNA–DNA hybridization data, allowed the new isolate to be identified as a strain of the species Methanosarcina mazei. The new isolate differed from the known representatives of this species in that it was resistant to alkylbenzene sulfonates and able to demethylate p-toluene sulfonate when grown on acetate.