Unialgal Strains Research Articles

Selected Case Studies on Fastidious Eukaryotic Microorganisms: Issues and Investigation Strategies

The concept of fastidious microorganisms currently found in scientific literature is mainly related to the difficulty of isolating/culturing/preserving bacteria. Eukaryotes are investigated much less in this respect, although they represent a fundamental part of the microbial world. Furthermore, not only isolation, but also identification and culturing (in the perspective of long-term preservation) should be considered key aspects often impacting on the study of fastidious microorganisms, especially in terms of preservation in culture collections and biotechnological exploitation. The present review aimed to investigate the current state of the art on fastidious eukaryotes, with special emphasis on the efforts to improve their isolation, identification, culturing and long-term preservation in culture collections practices. A few case studies focused on some fastidious eukaryotic microorganisms (including possible customized solutions to overcome specific issues) are also presented: isolation and preservation of slow-growing fungi, culturing of Haematococcus lacustris, isolation of unialgal strains of Cyanidiophytina (Rhodophyta), identification of Metschnikowia pulcherrima clade yeasts, isolation and preservation of Pyricularia species, preservation of Halophytophtora spp.

A New Deep-Water Epilithic Green Alga, Ulvella lacustris, from an Alpine Brackish Lake in Qinghai–Tibet Plateau

Ulvella species are widely distributed in ocean and freshwater habitats and from high latitudes to the tropics. However, no species of this genus have been found in the Qinghai–Tibetan Plateau. In the present study, five rock specimens were collected from the bottom of Qinghai Lake at a depth of about 15–28 m, from which three unialgal strains were isolated. These strains were characterized by prostrate pseudoparenchyma, irregular or radial branch filaments, and mono- or polystromatic discs or rosettes formed by consecutive filaments, with thalli that can grow up to 180 um in length, suggesting that they may be Ulvella species. Phylogenetic analysis based on 18S rDNA and tufA cpDNA sequences revealed that the isolates formed a strong branch among other Ulvella species and were closely related to Ulvella leptochaete and Ulvellawaernii. This result was also confirmed by neighbor-joining tree inference based on ITS2 secondary structure. Unlike other members of Ulvella, the isolated strain was characterized by its unique habitat, field morphology of thallus, size of vegetative cells, and number of pyrenoids. Based on these morphological differences, the phylogenetic analysis, and the comparison of ITS2 secondary structure with relative species, the strain isolated in this study was proposed to be a novel Ulvella sp. The alga was found on the bottom of brackish lakes on the Qinghai–Tibetan Plateau, which meant that our sampling range needed to be expanded further.

Different Growth and Sporulation Responses to Temperature Gradient among Obligate Apomictic Strains of Ulva prolifera.

The green macroalga Ulva prolifera has a number of variants, some of which are asexual (independent from sexual variants). Although it has been harvested for food, the yield is decreasing. To meet market demand, developing elite cultivars is required. The present study investigated the genetic stability of asexual variants, genotype (hsp90 gene sequences) and phenotype variations across a temperature gradient (10–30 °C) in an apomictic population. Asexual variants were collected from six localities in Japan and were isolated as an unialgal strain. The hsp90 gene sequences of six strains were different and each strain included multiple distinct alleles, suggesting that the strains were diploid and heterozygous. The responses of growth and sporulation versus temperature differed among strains. Differences in thermosensitivity among strains could be interpreted as the result of evolution and processes of adaptation to site-specific environmental conditions. Although carbon content did not differ among strains and cultivation temperatures, nitrogen content tended to increase at higher temperatures and there were differences among strains. A wide variety of asexual variants stably reproducing clonally would be advantageous in selecting elite cultivars for long-term cultivation. Using asexual variants as available resources for elite cultivars provides potential support for increasing the productivity of U. prolifera.

Integrative taxonomic description of the marine species Cocconeis crawfordii (Bacillariophyceae) based on unialgal strains

Integrative taxonomic description of the marine species Cocconeis crawfordii (Bacillariophyceae) based on unialgal strains

Integrative taxonomic description of two new species of the Cocconeis placentula group (Bacillariophyceae) from Korea based on unialgal strains

Cocconeis coreana and C. sijunghoensis are described as new based on micromorphological and molecular data. C. coreana is represented by five unialgal cultures from four different freshwater bodies, two from North Korea and three from South Korea. C. sijunghoensis is represented by two unialgal cultures from a brackish water body in North Korea. Except for one, all of the strains auxosporulated and showed an almost quadrupling of size in length and width. Morphologically, these species with their two different elliptical valves belong to the Cocconeis placentula group. The raphe valve has striae with uniseriate areolae continuing across a pronounced submarginal hyaline rim to the edge of the valve. The sternum valve has uniseriate dash-like areolae continuously from the valve face until the valve edge. Micromorphologically, these species possess two different open valvocopulae: only the raphe valvocopula has fimbriae; the sternum valvocopula has none. Based on p-distances of currently available DNA sequence data, i.e., rbcL and 18SV4, both species are pronouncedly different from the epitype strain of C. placentula, with C. coreana closest to the published molecular data of the strain UTEX FD23 named C. placentula from Iowa, USA, while C. sijunghoensis is closest but not the same as the published molecular data of strain D36_012, the epitype strain of C. placentula from Berlin, Germany. Based on scanning electron microscope observations, differentiating features are discussed concerning valvocopula fimbriae, central area, areolation of the sternum valve and on the raphe valve especially between the submarginal hyaline rim and edge. Key words: auxospores; cultures; micromorphology; molecular markers; morphometrics; 18SV4 & rbcL

The macroalgal culture collection in Kobe University (KU-MACC) and a comprehensive molecular phylogeny of macroalgae based on the culture strains

ABSTRACT The Kobe University Macroalgal Culture Collection (KU-MACC), as a section of the Kobe University Research Center for Inland Seas, distributes cultures of macroalgae to the public under the umbrella of the National BioResource Project of Ministry of Education, Culture, Sports, Science and Technology and Japan Agency for Medical Research and Development, in collaboration with the Microbial Culture Collection of the National Institute of Environmental Studies. KU-MACC principally holds macroalgae (Phaeophyceae, Rhodophyta and Ulvophyceae), but also holds some important related taxa of Schizocladiophyceae and Charophyceae. They are unialgal strains maintained as vegetative thalli and by cryo-preservation. Stock cultures are grown in culture chambers under various temperature conditions in transparent polypropylene vials containing Provasoli’s Enriched Seawater (PES) or PESI medium. The collection includes the “genome strains” of Ectocarpus siliculosus, Chondrus crispus, Chara braunii and their related stains. Depending on the phylogenetic group, DNA sequences of the nuclear rDNA ITS region, chloroplast rbcL, and mitochondrial cox3 genes are determined and used for taxonomy and as genetic tags. Based on the genetic data, molecular phylogenies of Ulvophyceae, Phaeophyceae and Rhodophyta are presented. In the rbcL molecular phylogeny of Ulvophyceae, due to rather limited coverage of culture strains, only members of Ulothricales, Ulvales and Bryopsidales are represented by KU-MACC strains, whereas 10 orders are recognized in the class. The molecular phylogeny of Phaeophyceae based on the cox3 gene showed a tree topology similar to the published multigene molecular phylogeny except for the position of Ishige okamurae, although the bootstrap supports of most nodes were low: Ishigeales branched near the base of Phaeophyceae next to Discosporangiales in the multigene molecular phylogeny, but in the cox3 tree, it showed a long branch and was sister to Fucales. In the molecular phylogeny of Rhodophyta, 14 orders are represented by KU-MACC strains covering about half of currently recognized Bangiophycean and Florideophycean orders.

Halophilic Algal Communities in Biological Soil Crusts Isolated From Potash Tailings Pile Areas

Mining potash for fertilizer produces vast amounts of highly saline waste that is deposited in potash tailings piles. Rainfall washes the salts out, affecting the surrounding ecosystems. Only salt-tolerant organisms occur close to the piles, since other species cannot survive in these hypersaline conditions. Halophytic plant communities around tailings piles have been well investigated. However, studies exploring the biodiversity of the space behind the vegetation line that separates suitable salinities for plant growth from hostile conditions are rare. On top of the tailings piles, only micro-organisms thrive. This study, for the first time, explored the microalgae and cyanobacteria in biocrusts that in-habit potash tailings pile areas between the vegetation line and the pile body. Two biocrust types were studied: young biocrusts at or close to the tailings piles, and established biocrusts situated in the near surroundings. The estimated abundance of green algae, cyanobacteria and diatoms was studied using a direct microscopy approach, followed by the isolation and establishment of unialgal strains and morphological species determinations. Soil environmental characteristics were analyzed. Soil samples showed ECSat (electrical conductivity) values that drastically exceeded the scales commonly used to describe soil salinity, indicating extremely saline conditions. Indeed, the isolate composition was shaped by soil salinity parameters. Soil ECSat of young biocrusts tended to be higher than in estab-lished biocrusts, and filamentous green algae were most abundant. In contrast, established biocrusts tended to have a lower ECSat and were mostly dominated by filamentous cyanobacteria. Algal and cyanobacteria isolate composition differed significantly in young and established biocrusts, although the species number did not. Some of the salt-tolerant algal strains are assumed to be candidates for the formation of artificial biocrusts on the surface of the tailings. Attempts to “green” the piles by the establishment of higher plants to trap rainwater and therefore reduce the salt-output are difficult to apply. Plants require a thick layer of substrate to reduce the salinity, and the substrate easily erodes on the steep slopes. However, microalgae isolated from potash tailings pile areas seem promising, since they can survive on a thinner substrate layer and are already adapted to these hypersaline conditions.

Suitable unialgal strains of Gracilariopsis chorda and Gracilaria vermiculophylla for hemagglutinin production

The selection of unialgal culture strains suitable as sources of algal hemagglutinins is one of the important factors for success in algal component production. To obtain suitable unialgal culture strains, several Gracilariopsis chorda and Gracilaria vermiculophylla strains were surveyed for hemagglutinating activity, relative growth rates, and fertility difficulty. Fertile tetrasporophyte samples of naturally occurring G. chorda and G. vermiculophylla were collected at each of three different collection sites around Shikoku Island in southwest Japan. Unialgal culture strains were started from isolated tetraspores obtained from each naturally occurring plant. The hemagglutinating activities in algal extracts of the three G. chorda strains (3100–7000 units mg−1) were higher than those of the three G. vermiculophylla strains (920–980 units mg−1). The daily growth rates of the G. chorda strains (8.0–13.8% day−1 at 22 °C) were higher than those of the G. vermiculophylla strains (4.1–4.4% day−1 at 22 °C). Among the unialgal culture strains tested, the one started from isolated tetraspores from G. chorda growing in the Katsuura River had the highest hemagglutinating activity and relative growth rate. This strain also did not become fertile even after a period of 3 years of culture with aeration at 22 °C, in a 14-h light–10-h dark cycle at 60 μmol photons m−2 s−1. Thus, the unialgal culture strain started from isolated tetraspores from G. chorda growing in the Katsuura River seems to be a useful source for hemagglutinin production.

Symbiotic chlamydomonads in algological department collection of BIN RAS

General information on the unialgal strains of green monads, mass-developing in symbiosis with other eukaryotic organisms from North-West Russia, is presented. The selected strains are maintained in the Algological Department of Komarov Botanical Institute RAS. The previous collection acronim LABIK – collection of algae of Laboratory Algology of Komarov Botanical Institute is suggested to remain. Topics on the isolation and strain identification difficulties of algae, living together with other organisms, as well as, their partners relations and perspectives of biotechnological usage are concidered.

Gomphonella olivacea (Bacillariophyceae) – a new phylogenetic position for a well-known taxon, its typification, new species and combinations

Background and aims – Within the project “German Barcode of Life – Diatoms” common diatoms of German waters were routinely isolated and cultivated. In order to understand the taxonomy and phylogeny of the genus Gomphonema, one of the most common taxa of Central Europe, known currently either under the name Gomphonema olivaceum in Europe or Gomphoneis olivacea in America, was studied.
 Methods – Twenty unialgal strains were established from five different water bodies in Germany and one from Lake Balaton, Hungary, which supplied molecular data (18S V4 and rbcL) besides morphometric and ultrastructural data. In addition, on eight populations from different water bodies including the type from Denmark, morphometric and micromorphological studies by light and scanning electron microscopy were performed.
 Key results – Molecular and micromorphological data show that the target taxon neither belongs to Gomphonema Ehrenb. nor to Gomphoneis Cleve. By reinstating the genus name Gomphonella Rabenh., the nomenclatural and taxonomic enigma of this taxon is solved, and with the presentation of the type by Hornemann the authorship of the epithet is clarified. Molecular data for the unialgal strains and several environmental clones show that there is more diversity in the Gomphonella olivacea clade than can be identified morphologically. In addition, the establishment of the new species Gomphonella coxiae and Gomphonella acsiae is supported. The molecular data classified Gomphonella species as belonging to the Cymbellales but not to the Gomphonemataceae. In addition, molecular data put Gomphoneis tegelensis R.Jahn & N.Abarca also into Gomphonella. In order to make the genera Gomphoneis and Gomphonema monophyletic, their astigmate members are transferred to Gomphonella.
 Conclusions – The results clarify that the gomphonemoid outline is not restricted to the family Gomphonemataceae but seem to be distributed across the entire order Cymbellales. This is shown in this paper for the revived genus Gomphonella, which contains the astigmate group of Gomphoneis and Gomphonema besides the longly disputed G. olivacea. Only a polyphasic approach, combining molecular and micromorphological data for taxonomy, nomenclatural evaluation, and observations from clonal cultures can reveal the full intricacies of evolutionary relations.

Geosmin-producing Species of Coelosphaerium (Synechococcales, Cyanobacteria) in Lake Shinji, Japan

In Lake Shinji, Japan, periodic outbreaks of musty odour have occurred since mid-May 2007. Although the substance responsible for the odour was identified as geosmin, the odour-producing organism was unknown. We cultivated an axenic unialgal strain and determined that a species of Coelosphaerium (Synechococcales) was responsible for the production of geosmin in Lake Shinji. Our analysis was conducted using gas chromatography/mass spectrometry to determine the odorous compound. To determine the algae species, it was observed by optical microscopy to describe its morphological characteristics and the polymerase chain reaction was used to characterise the nucleotide sequence of the 16S rRNA gene and the 16S-23S rRNA internal transcribed spacer region. In addition, we explored the relationship between the number of cells of the Coelosphaerium sp. and the concentration of geosmin. In conclusion, geosmin, the cause of the musty odour in Lake Shinji in autumn 2009, was produced by Coelosphaerium sp., and to our knowledge, this is the first report of a geosmin-producing species in the family Coelosphaeriaceae.

Phenotypic and genotypic validation of the rare species Sphaerospermopsis eucompacta comb. nov. (Nostocales, Cyanobacteria) isolated from China

:The heterocystous cyanobacterium Anabaena eucompacta R. Li & M. Watanabe was found in China, and two unialgal strains were isolated. The species was characterized by its twisted and tight spirals and the relative position of spherical akinetes to heterocysts (adjacent to one or both sides of heterocysts). However, the taxonomic and phylogenetic position of the species remained to be characterized because of the lack of molecular data. In this study, we used a polyphasic approach, based on both morphological and molecular characters (sequences of 16S rRNA and 16S-23S rRNA), to evaluate its taxonomic position. Our results showed that A. eucompacta was a distinct species and that it should be transferred to the recently established genus Sphaerospermopsis as S. eucompacta (R. Li & M. Watanabe) X. Li & R. Li comb. nov.

Biomass and neutral lipid production in geothermal microalgal consortia.

Recently, technologies have been developed that offer the possibility of using algal biomass as feedstocks to energy producing systems – in addition to oil-derived fuels (Bird et al., 2011, 2012). Growing native mixed microalgal consortia for biomass in association with geothermal resources has the potential to mitigate negative impacts of seasonally low temperatures on biomass production systems as well as mitigate some of the challenges associated with growing unialgal strains. We assessed community composition, growth rates, biomass, and neutral lipid production of microalgal consortia obtained from geothermal hot springs in the Great Basin/Nevada area that were cultured under different thermal and light conditions. Biomass production rates ranged from 39.0 to 344.1 mg C L−1 day−1. The neutral lipid production in these consortia with and without shifts to lower temperatures and additions of bicarbonate (both environmental parameters that have been shown to enhance neutral lipid production) ranged from 0 to 38.74 mg free fatty acids (FFA) and triacylglycerols (TAG) L−1 day−1; the upper value was approximately 6% of the biomass produced. The higher lipid values were most likely due to the presence of Achnanthidium sp. Palmitic and stearic acids were the dominant free fatty acids. The S/U ratio (the saturated to unsaturated FA ratio) decreased for cultures shifted from their original temperature to 15°C. Biomass production was within the upper limits of those reported for individual strains, and production of neutral lipids was increased with secondary treatment. All results demonstrate a potential of culturing and manipulating resultant microalgal consortia for biomass-based energy production and perhaps even for biofuels.

A novel genus Ammassolinea gen. nov. (Cyanobacteria) isolated from sub-tropical epipelic habitats.

A new monospecific genus of an epipelic, filamentous oscillatorean Phormidium-like cyanobacterium is established. The genus was first discovered from a pond at Fiddlers Ridge Drive, Jacksonville, Florida, USA and subsequently isolated into uni-algal strains. During the process of cultivation, a combination of morphological, ultrastructural and molecular evidence supports the erection of a novel genus. Based on apomorphies which differ from related taxa such as heap-like colony formation, ultrastructural properties and molecular uniqueness, we propose the generic epithet Ammassolinea (sand-dwelling filaments).

Characterization of the new microalgal strains, Oogamochlamys spp., and their potential for biofuel production

Isolation of novel lipid-producing microalgae that can easily grow under photoautotrophic conditions is promising for biofuel production. In this study, we isolated indigenous microalgal strains and screened their lipid-producing activity. Freshwater supplemented with several nutrients was used for the cultivation of indigenous microalgae to minimize enrichment bias. Photoautotrophic cultivation in microplates resulted in 388 unialgal strains, and 59 strains of them were stained by Nile Red. Phylogenetic studies based on 18S ribosomal DNA suggested that these belonged to 6 genera including Chlorella, Scenedesmus, Monoraphidium, Coccomyxa, Macrochloris, and Oogamochlamys. Considering the cellular lipid content and biomass yield under photoautotrophic conditions, Oogamochlamys spp. IKE31 and IKE32 show promise for biofuel production. These strains are rich in unsaturated fatty acids, which implies that they may also be useful as feed for animals and aquaculture species. Lipid productivity of the strains was dramatically increased by phosphate limitation, which may be beneficial for industrial applications.

The phylogeny of Heterocapsa sp. (Dinophyceae) isolated from the south coast of Iran during a Cochlodinium polykrikoides bloom

The genus Heterocapsa Stein is a relatively small armoured dinoflagellate. Because it has few differences in morphological characteristics among its species, species identification is difficult. In this study, small Heterocapsa sp. cells associated with a dense bloom of Cochlodinium polykrikoides Margalef were collected from the south-east coast of Iran. While Heterocapsa sp. was isolated, C. polykrikoides could not be isolated by single cell or serial dilution methods. Thus, a unialgal strain of Heterocapsa sp. was used for molecular analysis and species identification. In order to carry out phylogenetic analysis, rDNA was extracted and large subunit domains of D1-D3 were sequenced. Similar sequences of other geographical strains were selected from GenBank and compared with the Iranian species. The Iranian strain was aligned with Heterocapsa spp. Phylogenetic analysis of maximum likelihood and neighbor-joining demonstrated that Heterocapsa sp. does not form a monophyletic group. Morphological characteristics confirmed molecular results. This is the first record of Heterocapsa isolation from Iranian coastal waters.

Biotransformation of monoterpenes by immobilized microalgae

This paper reports the biotransformation of carvone, limonene, β-pinene, thymol, and linalool using whole-cell-immobilized microalgal strains isolated from paddy fields of Iran. The strains was recognized by morphological characterization and assigned according to amplified 16S/18S rRNA genes by PCR. Ten unialgal strains including Chlorella, Oocystis, Chlamydomonas, and Synechococcus were immobilized in calcium alginate beads. After a 24-h incubation with substrates, characterization and identification of biotransformation products were done by GC/MS. None of the isolated immobilized microalgae converted β-pinene. In contrast, most of these strains biotransformed carvone and limonene to the related compounds. Some strains only reduced the C = C double bond to yield the dihydrocarvone isomers while others reduced the ketone to give the dihydrocarveol. The transformation ratio showed that Oocystis sp. MCCS 033 and Synechococcus sp. MCCS 035 produced dihydrocarvone isomers with the highest efficiency. Furthermore, limonene was converted into a mixture of five corresponding products and the maximum yield was 52.1% for carvone, the bioconverted product. Only one strain, Synechococcus sp. MCCS 034, oxidized thymol, and the product obtained from thymol was thymoquinone. Also, linalooloxide isomers and dihydrolinalool were obtained from linalool, and finally dihydrolinalool was the main product. These results showed a novel conversion pathway of linalool-forming dihydrolinalool.

OPTIMAL GROWTH CONDITIONS AND ANTIOXIDATIVE ACTIVITIES OF CYLINDROTHECA CLOSTERIUM (BACILLARIOPHYCEAE)(1).

We isolated the unialgal strain of Cylindotheca closterium (Ehrenb.) Reimann et J. C. Lewin and produced an axenic strain using an antibiotic cocktail of enriched f/2 artificial seawater medium. The optimal growth conditions were estimated under 27 different combinations of temperature, salinity, and nutrients, and mass culture was performed based on the best specific growth conditions. Its antioxidant activities were determined from the extracts of methanol, water, and enzymes (proteases and carbohydrases). The maximum specific growth rate (μmax ) varied from 0.63 to 0.97 · d(-1) . The maximum cell density was 7.20 × 10(4) cells · mL(-1) , while the μmax was 0.82 · d(-1) in culture conditions of 20°C, 30 psu (practical salinity unit), and "F" nutrient concentrations on day 10 of the culture period. The scavenging rates for 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical were 72.5% and 69.4% from Viscozyme and methanol extracts, respectively. The enzymatic extracts of C. closterium prepared by the hydrolyses of Amyloglucosidase (AMG) and Viscozyme showed 45.8% and 45.5% nitric-oxide-scavenging rates, slightly lower than the activity of alpha-tocopherol (α-tocopherol) but similar to butylated hydroxytoluene (BHT). The extract from methanol and water showed 44.8% and 44.4% scavenging rates, statistically similar with BHT. The metal-chelating activities of the Kojizyme, Alcalase, methanol, Viscozyme, and Neutrase extracts were 67.1, 53.9, 53.2, 52.1, and 50.2 %, respectively, five to six times higher than the commercial antioxidants. The AMG, Viscozyme, and Neutrase extracts showed a remarkable linoleic acid peroxidation inhibition, which was higher than BHT and statistically similar with α-tocopherol.

Benthic cyanobacteria (Oscillatoriaceae) that produce microcystin-LR, isolated from four reservoirs in southern California

Cyanobacteria that produce the toxin microcystin have been isolated from many parts of the world. Most of these organisms are planktonic; however, we report on several microcystin-producing benthic filamentous cyanobacterial isolates from four drinking-water reservoirs in southern California (USA): Lake Mathews, Lake Skinner, Diamond Valley Lake (DVL), and Lake Perris. Some samples of benthic material from these reservoirs tested positive for microcystin by an ELISA tube assay, and all the positive samples had in common a green filamentous cyanobacterium 10–15 μm in diameter. Seventeen unialgal strains of the organism were isolated and tested positive by ELISA, and 11 cultures of these strains were found to contain high concentrations of microcystin-LR (90–432 μg L −1). The cultures were analyzed by protein phosphatase inhibition assay (PPIA) and HPLC with photodiode array detector (PDA) or liquid chromatography/mass spectrometry (LC/MS). Microcystin per unit carbon was determined for six cultures and ranged from 1.15 to 4.15 μg mg −1 C. Phylogenetic analysis of four cultures from Lake Skinner and DVL using cyanobacterial-specific PCR and sequencing of the partial 16S rRNA gene suggested the highest similarity to an unidentified cyanobacterium in the oscillatoriales, and to a Phormidium sp. Morphologically, some of the isolates were similar to Oscillatoria, and others resembled Lyngbya. The significance of these organisms lies in the relative scarcity of known toxin producers among freshwater benthic cyanobacteria, and also as a source of cell-bound microcystin in these reservoirs.

N 2-fixation and complementary chromatic adaptation in non-heterocystous cyanobacteria from Lake Constance

Non-heterocystous, mostly filamentous cyanobacteria were isolated from the crust of stones, from the periphyton of two macrophytes from the littoral zone and from the pelagic environment of Lake Constance. All isolates were cultivated as unialgal strains. DNA analysis by restriction fragment length polymorphism with the psbA gene probe revealed high genetic diversity among the strains from the littoral zone. For all genotypes, the occurrence of the nifH gene encoding a nitrogenase subunit and of genes encoding subunits of phycoerythrin and phycocyanin were tested by Southern blot hybridization. In addition, the isolates were investigated for their ability for complementary chromatic adaptation (CCA) and for anaerobic N 2-fixation. With respect to these characteristics, all cyanobacteria included in this study were assigned to four different types: (1) strains without the capability to fix N 2 or to perform CCA of the group III type (CCA III); (2) strains which show both features; (3) strains with the ability to fix nitrogen, but that do not show any CCA III; and (4) strains that produce phycoerythrin, but without the capacity for CCA III or N 2-fixation. By examining the frequency distribution of isolates, these types were shown to prefer different habitats. While cyanobacterial strains capable of N 2-fixation, but without CCA III, were mainly obtained from stone crusts in the supralittoral zone, those with the potential for N 2-fixation as well as for CCA III were largely isolated from submersed macrophytes. Cyanobacteria that produce phycoerythrin, but do not perform CCA III or N 2-fixation, were found in the pelagic zone only.