Use of filamentous cyanobacteria for biodegradation of organic pollutants

Volume 61, no. 1, p. 234: the corresponding author footnote should read as follows: * Corresponding author. Present address: Center for Risk Management, Oak Ridge National Laboratory, Oak Ridge, TN 37830. Phone: (615) 241-6013. Fax: (615) 574-9887. [This corrects the article on p. 234 in vol. 61.].     

The production of biomass and phycobiliprotein pigments in filamentous cyanobacteria: the impact of light and carbon sources

Cyanobacteria are recognized as producers of bioactive substances and phycobiliproteins, whose medicinal and functional food properties have led to increased interest in recent years. In the present study, the biomass production and phycobiliprotein content in cyanobacterial strains belonging to Anabaena, Nostoc and Spirulina genera were investigated under the conditions of continuous illumination and mixotrophic nutrition. The results showed that biomass production was strongly stimulated by continuous light in Spirulina strains (4.5-fold), and by organic carbon sources in N₂-fixing strains (2.1–2.8-fold). The strategy of cells to accumulate primarily blue pigment phycocyanin and bluish green allophycocyanin was revealed under tested conditions. Furthermore, in the case of Spirulina S1 grown with glycerol, the culture medium became dense and changed its colour to pink, which may indicate the release of compounds including pigment(s) outside the cell, the phenomenon that seem to be rare among cyanobacteria. Moreover, under continuous light, in this strain the highest biomass level of 4.0 mg/mL was achieved, wherein phycocyanin and allophycocyanin content was increased 12- and 16-fold, respectively, which indicates the high potential of this strain for further investigation.     

Use of filamentous cyanobacteria for biodegradation of organic pollutants.

Biodegradation is increasingly being considered as a less expensive alternative to physical and chemical means of decomposing organic pollutants. Pathways of biodegradation have been characterized for a number of heterotrophic microorganisms, mostly soil isolates, some of which have been used for remediation of water. Because cyanobacteria are photoautotrophic and some can fix atmospheric nitrogen, their use for bioremediation of surface waters would circumvent the need to supply biodegradative heterotrophs with organic nutrients. This paper demonstrates that two filamentous cyanobacteria have a natural ability to degrade a highly chlorinated aliphatic pesticide, lindane (gamma-hexachlorocyclohexane); presents quantitative evidence that this ability can be enhanced by genetic engineering; and provides qualitative evidence that those two strains can be genetically engineered to degrade another chlorinated pollutant, 4-chlorobenzoate.     

Perforation patterns in the peptidoglycan wall of filamentous cyanobacteria

Ultrastructural studies were made on seven genera of filamentous cyanobacteria from Sections III and IV (in the Rippka classification): Oscillatoria limosa Ag. ex Gomont, Spirulina subsalsa Turp. ex Gomont, Crinalium epipsammum Crow, Nostoc commune Vaucher ex Born. et Flah., Anabaena variabilis Kützinger ex Born. et Flah., Arthrospira maxima Stizenb. ex Gomont, Arthrospira platensis Stizenb. ex Gomont, and Cyanospira rippkae Florenzano. Perforation types and their distribution in the peptidoglycan layer observed in Crinalium epipsammum Crow were similar to those in Oscillatoria limosa and two Arthrospira strains. In Cyanospira and Anabaena no junctional perforations in the peptidoglycan wall layer were observed; however, intracellular perforations were documented. Except for Spirulina , the perforations were organized in rows (up to 10) placed on both sides of the cross wall. In the Spirulina cross sections, perforations were found only in the thickened, inner part of the cell wall. The perforation patterns in Spirulina and Arthrospira strains were clearly different and justify the separation into different genera.     

Characterization and cloning of extrachromosomal DNA from filamentous cyanobacteria

Abstract Physical maps of cryptic plasmids from the filamentous cyanobacteria Anabaena variabilis PCC7118 (pGL1: 3.6 MDa), Nostoc PCC6705 (pGL2: 2.6 MDa) and Plectonema PCC6306 (pGL3: 0.95 MDa) were generated. Selectable markers were introduced onto pGL2 and pGL3 by fusing them to the vector pBR328, using their single restriction sites for Cla I. The recombinant plasmids generated were characterised with respect to the orientation of the insert and the single sites for restriction endonucleases which they possess. The stability of pGL1 and of the two recombinant plasmids in culture was investigated and a method for isolating larger cyanobacterial plasmids (> 20 MDa) was devised.     

Probing environmental DNA reveals circum-Baltic presence and diversity of chlorophyll a/b-containing filamentous cyanobacteria (genus Prochlorothrix)

Cyanobacteria that possess phycobilisomes, light-harvesting antenna, have been well studied. In contrast, more rare green cyanobacteria (four genera/five species) that instead make use of chlorophyll–protein complex are poorly studied. In particular, the genus Prochlorothrix is represented by a small environmental DNA database and reports of only two cultured species from Northern Europe. In this work, marine and freshwater habitats of Northwestern Russia were investigated. PCR with Prochlorothrix 16S rRNA gene specific primers, FISH analysis with a Prochlorothrix 16S rRNA-targeted probe, Prochlorothrix culture isolation, and phylogenetic analysis of Prochlorothrix diversity were carried out. We identified Prochlorothrix 16S rDNA in samples from the St. Petersburg region and corroborated this finding by FISH. Attempts to isolate PCR- and FISH-detected Prochlorothrix strains were unsuccessful. Phylogenetic analysis revealed that the Prochlorothrix 16S rDNA sequences identified were very similar and formed a single cluster with high bootstrap support. Some of these sequences represent environmental strains of the species Prochlorothrix hollandica and P. scandica, while the others belong to new Prochlorothrix species or even to a new Prochlorothrix-related genus. Our results suggest a broader distribution and greater diversity in Prochlorothrix than previously thought.     

Survey of extrachromosomal DNA found in the filamentous cyanobacteria.

Cleared lysates of 13 species of filamentous cyanobacteria were examined for the presence of extrachromosomal DNA by using agarose gel electrophoresis and ethidium bromide staining. Seven of the 13 species contained extrachromosomal covalently closed circular DNA, and all but 1 species contained multiple elements. There was no correlation between the presence of extrachomosomal DNA and either the range of metabolic activities found in the cyanobacteria or the differentiated cell types or structures elaborated by the morphologically complex filamentous cyanobacteria.     

Molecular cloning and characterization of hetR genes from filamentous cyanobacteria

HetR, a serine type protease, plays an important role in heterocyst differentiation in filamentous cyanobacteria. We isolated and sequenced the hetR genes from different heterocystous and filamentous nonheterocystous cyanobacteria. The hetR gene in the heterocyst forming Anabaena variabilis ATCC 29413 FD was interrupted by interposon mutagenesis (mutant strain WSIII8). This mutant does not form heterocysts and shows no diazotrophic growth under aerobic conditions. However, under anaerobic N(2)-fixing conditions, the WSIII8 cells are able to grow, and high nitrogenase (Nif2) activity is detectable. Nif2 expression was demonstrated in each vegetative cell of the filament by immunolocalization 4 h after nitrogen step-down.     

Effects of tropical solar radiation on the motility of filamentous cyanobacteria

Abstract The effects of tropical solar radiation on the motility of the cyanobacteria Anabaena variabilis, Oscillatoria tenuis and two strains of Phormidium uncinatum were studied in Ghana (4.30°N). The percentages of motile filaments were drastically reduced by unfiltered solar radiation. Covering the organisms with various long pass or band pass filters (WG320, GG395 or UG5) revealed that the UV-B, UV-A and visible light components of the solar spectrum were all effective in impairing motility in these organisms. Only partial recovery was observed and only after short exposure times.     

Generation of reactive oxygen species upon red light exposure of cyanobacteria from Roman hypogea

Subterranean archaeological sites in Rome (Italy) are threatened by phototrophic biofilms predominated by cyanobacteria and associated microorganisms. They damage the frescoes, mortar, marble, and tufa rock wherever artificial lighting is installed. During the past two decades, the conservation strategies have evolved gradually; rather than restricting the illumination time and intensity, the latest approach is to use strong light to reduce their growth. Since cyanobacterial cells are abundant in phycobilisomes and chlorophyll a, which produce reactive oxygen species (ROS) upon irradiation, strong red light (620–650 nm) was applied to generate high amount of ROS in a rate beyond the quenching capacity of the organism. After 25 h of irradiation, the photosystem II quantum yields of seven cyanobacterial isolates in culture were reduced by 65–94%. Conversely, blue light (460–480 nm) promoted photosystem II activity by up to 35%. δ-Aminolevulinic acid (D-ALA) was introduced to enhance the treatment, as it can be transformed into protochlorophyllide by cyanobacteria and then excited by red light to generate ROS inside the cells. Since the natural photosynthetic pigments as well as the endogenous protochlorophyllide exist only within the cyanobacterial cells, they are unlikely to contaminate or damage the underlying stone substrata. Electron spin resonance spectroscopy confirmed that D-ALA treatment caused the formation of ROS; spin trap experiments indicated that radicals were produced in the system.     

Occurrence of facultative anoxygenic photosynthesis among filamentous and unicellular cyanobacteria.

Eleven of 21 cyanobacteria strains examined are capable of facultative anoxygenic photosynthesis, as shown by their ability to photoassimilate CO2 in the presence of Na2S, 3-(3,4-dichlorophenyl)-1,1-dimethylurea and 703-nm light. These include different cyanobacterial types (filamentous and unicellular) of different growth histories (aerobic, anaerobic, and marine and freshwater). Oscillatoria limnetica, Aphanothece halophytica (7418), and Lyngbya (7104) have different optimal concentrations of Na2S permitting CO2 photoassimilation, above which the rate decreases: 3.5, 0.7, and 0.1 mM, respectively. In A. halophytica, for each CO2 molecule photoassimilated two sulfide molecules are oxidized to elemental sulfur, which is excreted from the cells.The ecological and evolutionary significance of anoxygenic photosynthesis in the cyanobacteria is discussed.     

Antioxidant activity and phenolic profile in filamentous cyanobacteria: the impact of nitrogen

In the present study, ethanolic extracts of ten cyanobacterial strains cultivated under different nitrogen conditions were assessed for the phenolic content and antioxidant activity. The amount of detected phenolic compounds ranged from 14.86 to 701.69 μg g^?1 dry weight (dw) and HPLC-MS/MS analysis revealed gallic acid, chlorogenic acid, quinic acid, catechin, epicatechin, kaempferol, rutin and apiin. Only catechin, among the detected phenolics, was present in all the tested strains, while quinic acid was the most dominant compound in all the tested Nostoc strains. The results also indicated the possibility of increasing the phenolic content in cyanobacterial biomass by manipulating nitrogen conditions, such as in the case of quinic acid in Nostoc 2S7B from 70.83 to 594.43 μg g^?1 dw. The highest radical scavenging activity in DPPH assay expressed Nostoc LC1B with IC₅₀ value of 0.04?±?0.01 mg mL^?1, while Nostoc 2S3B with IC₅₀ =?9.47?±?3.61 mg mL^?1 was the least potent. Furthermore, the reducing power determined by FRAP assay ranged from 8.36?±?0.08 to 21.01?±?1.66 mg AAE g^?1, and it was significantly different among the tested genera. The Arthrospira strains exhibited the highest activity, which in the case of Arthrospira S1 was approximately twofold higher in comparison to those in nitrogen-fixing strains. In addition to this, statistical analysis has indicated that detected phenolics were not major contributor to antioxidant capacities of tested cyanobacteria. However, this study highlights cyanobacteria of the genera Nostoc, Anabaena, and Arthrospira as producers of antioxidants and phenolics with pharmacological and health-beneficial effects, i.e., quinic acid and catechin in particular.     

土壤丝状蓝藻在荒漠治理中的作用研究

土地荒漠化是当前全球性的重要环境问题之一。我国是世界上荒漠化面积较大,危害最严重的国家之一,全国荒漠和荒漠化土地面积2·782×106km2,约占国土陆地面积的三分之一。荒漠化土壤常存在区域性结皮现象,这种结皮对改善荒漠化土壤具有极其重要的作用。导致结皮的产生有物理的、化学的和生物的因素,其中以生物结皮最具生态意义,而生物结皮又分藻结皮、苔藓结皮和地衣结皮。藻类是荒漠中的先锋植物,尤其是一些抗逆性很强的蓝藻,特别是丝状蓝藻在荒漠土壤中具有更强的拓殖作用。本文从丝状蓝藻在生理生态上的优势综合介绍其在荒漠治理中不可替…     

Plasmid content and homology of 16 strains of filamentous,nonheterocystous cyanobacteria

We have developed several strain-specific, rapid, small-scale plasmid isolation procedures in order to characterize the plasmid profiles of 16 filamentous, nonheterocstous cyanobacteria. At least one distinct plasmid was found in eight strains, with seven of these containing two or more different plasmids. Eight strains were found to be without plasmid DNA. Both the large, 12.9 kb, and the small, 1.6 kb, plasmids fromPlectonema boryanum 581 were isolated, purified, and cloned. Southern blots of plasmid DNAs from the eight strains were probed with these cloned DNAs and also with ultra-pure plasmid DNA fromPhormidium liridum 426. Four strains ofP. boryanum (485, 581, 594, 1542) andP. luridum 426 have identical plasmid profiles, and plasmid homology is extensive.     

Plasmid distribution among unicellular and filamentous cyanobacteria: Occurrence of large and mega-plasmids

Abstract The plasmid content of 7 unicellular and 4 filamentous cyanobacterial strains has been analyzed. All strains were found to carry small plasmids while some strains harbor large plasmids up to approx. 400 kb. In addition, at least one megaplasmid of about 1000 kb was detected in the unicellular strain Synechococcus PCC7942. In the filamentous strain Calothrix PCC7601, 2 different distribution patterns of plasmids were observed in different subcultures, suggesting the presence of mobile DNA elements in this strain.     

Development of a biofertilizer based on filamentous nitrogen-fixing cyanobacteria for rice crops in Chile

The purpose of this study was to develop a biofertilizer based on filamentous nitrogen-fixing cyanobacteria selected from rice fields and to generate a technological package compatible with its use for the rice crop in Chile. Thirty-four Chilean rice fields, located between Maule and BioBío regions, were sampled during the 1998/1999 and 1999/2000 growing seasons. A total of 9 species and 3 varieties of cyanobacteria were found, and the nitrogen fixation rate under laboratory conditions was determined for 6 of them. Only 4 were used for the small-scale production of a biofertilizer, which was assayed in field trials. To check the efficiency of the biofertilizer during the rice crop, the nitrogen fixation rates in soil samples were estimated. Additionally, the biofertilizer application efficiency was tested in combination with nitrogen synthetic fertilizer, in rates that were previously established in field trials. Biofertilization allowed a decrease of up to 50% in the use of nitrogen synthetic fertilizer (50 kg N ha⁻¹), resulting in the same grain yield (7.4 t ha⁻¹) and quality in relation to the fertilized control. The use of biofertilizers based on local strains of cyanobacteria shows promise to increase nitrogen use efficiency in rice.     

Diversity of microcystin genotypes among populations of the filamentous cyanobacteria Planktothrix rubescens and Planktothrix agardhii

Microcystins (MCs) are toxic heptapeptides that are produced by filamentous cyanobacteria Planktothrix rubescens and Planktothrix agardhii via nonribosomal peptide synthesis. MCs share a common structure cyclo (-D-Alanine(1)-L-X(2)- D-erythro-beta-iso-aspartic acid(3)-L-Z(4)-Adda(5)-D-Glutamate(6)- N-methyl-dehydroalanine(7)) where X(2) and Z(2) are variable L-amino acids in positions 2, 4 of the molecule. Part of the mcyB gene (1,451 bp) that is involved in the activation of the X(2) amino acid during MC synthesis was sequenced in 49 strains containing different proportions of arginine, homotyrosine, and leucine in position 2 of the MC molecule. Twenty-five genotypes were found that consisted of eight genotype groups (A-H, comprising 2-11 strains) and 17 unique genotypes. P. rubescens and P. agardhii partly consisted of the same mcyB genotypes. The occurrence of numerous putative recombination events that affected all of the genotypes can explain the conflict between taxonomy and mcyB genotype distribution. Genotypes B (homotyrosine and leucine in X(2)) and C (arginine in X(2)) showed higher nonsynonymous/synonymous (d(N)/d(S)) substitution ratios implying a relaxation of selective constraints. In contrast, other genotypes (arginine, leucine, homotyrosine) showed lowest d(N)/d(S) ratios implying purifying selection. Restriction fragment length polymorphism (RFLP) revealed the unambiguous identification of mcyB genotypes, which are indicative of variable X(2) amino acids in eight populations of P. rubescens in the Alps (Austria, Germany, and Switzerland). The populations were found to differ significantly in the proportion of specific genotypes and the number of genotypes that occurred over several years. It is concluded that spatial isolation might favour the genetic divergence of microcystin synthesis in Planktothrix spp.