Assessment of the potential of Nostoc strains from the Pasteur Culture Collection for the production of polysaccharides of applied interest

The exocellular polysaccharides released during thephotoautotrophic growth by 25 Nostoc strainsbelonging to the Pasteur Culture Collection (PCC) wereinvestigated with regard to their chemical andrheological properties in order to assess theirpotential for the production of polysaccharides ofindustrial interest.All the released polysaccharides (RPSs) were complexanionic heteropolymers, composed of a number ofmonosaccharides ranging from six to nine, alwaysincluding glucose and fucose, and most frequentlygalactose. Ribose, rarely reported to be present incyanobacterial RPSs, was found in seven polymers. TheRPSs were shown to possess an anionic charge due tothe presence of uronic acids and in most cases also ofpyruvil and sulphate groups. Moreover, a large numberof polymers showed the presence of significant amountsof constituents such as acetyl groups, peptidicmoieties and deoxysugars, that may contribute to thehydrophobicity of the macromolecules. The viscositydependence on the shear rate of aqueous solutions ofthe RPSs was, in three cases, comparable with that ofxanthan gum, and one RPS showed a very satisfactorystability of the viscosity over a wide range of pH,temperature and salinity values. The most promisingPCC Nostoc strains for the production of RPSsfor specific applications were thus singled out, basedon the characteristics of their polymers.     

Heavy metal sorption by released polysaccharides and whole cultures of two exopolysaccharide-producing cyanobacteria

The metal removal capacity of cultures of two capsulated, exopolysaccharide-producing cyanobacteria, Cyanospira capsulata and Nostoc PCC7936, were tested using copper (II) as the model metal. C. capsulata cultures removed the greatest amount of copper, with a maximum per unit of biomass (q _max) of 115.0±5.1 mg copper g⁻¹ of protein, compared with 85.0±3.2 removed with Nostoc PCC7936 cultures. Water solutions of pure polysaccharides (RPSs) released into the culture medium by C. capsulata and Nostoc PCC7936 achieved q _max values of 20.2±0.8 mg g⁻¹ copper per polysaccharide dry weight with C. capsulata RPS and 11.0±1.5 mg g⁻¹ with Nostoc PCC7936 RPS. Cultures of the two cyanobacteria also removed Zn (II) and Ni (II), in both single-metal systems and in multimetal systems with Cu; in the various single-metal systems more copper was removed than Zn or Ni, while in the multimetal systems a smaller amount of each individual metal was removed but the overall amount of all metal ions sorbed or the amount of copper sorbed in the copper-only system was almost the same with C. capsulata, and slightly higher with Nostoc PCC7936.     

Sheathless mutant of Cyanobacterium Gloeothece sp. strain PCC 6909 with increased capacity to remove copper ions from aqueous solutions

The cyanobacterium Gloeothece sp. strain PCC 6909 and its sheathless mutant were tested for their abilities to remove copper ions from aqueous solutions, with the aim of defining the role of the various outermost polysaccharidic investments in the removal of the metal ions. Microscopy studies and chemical analyses revealed that, although the mutant does not possess a sheath, it releases large amounts of polysaccharidic material (released exocellular polysaccharides [RPS]) into the culture medium. The RPS of the wild type and the mutant are composed of the same 11 sugars, although they are present in different amounts, and the RPS of the mutant possesses a larger amount of acidic sugars and a smaller amount of deoxysugars than the wild type. Unexpectedly, whole cultures of the mutant were more effective in the removal of the heavy metal than the wild type (46.3 +/- 3.1 and 26.7 +/- 1.5 mg of Cu(2+) removed per g of dry weight, respectively). Moreover, we demonstrated that the contribution of the sheath to the metal-removal capacity of the wild type is scarce and that the RPS of the mutant is more efficient in removing copper. This suggests that the metal ions are preferably bound to the cell wall and to RPS functional groups rather than to the sheath. Therefore, the increased copper binding efficiency observed with the sheathless mutant can be attributed to the release of a polysaccharide containing larger amounts and/or more accessible functional groups (e.g., carboxyl and amide groups).     

Effect of <Emphasis Type="Italic">Nostoc</Emphasis> sp. on soil characteristics,plant growth and nutrient uptake

The arid desert area is expanding by 6 million ha year⁻¹. Nostoc, a terrestrial cyanobacterium, is known as a pioneer organism which can photosynthesize, fix atmospheric nitrogen, and secrete polysaccharides. In order to explore the potential of Nostoc as a countermeasure to soil desertification, we have investigated the effects of Nostoc on the chemical and physical properties of soil and on plant growth in outdoor and laboratory experiments. The effect of Nostoc on soil properties, when applied on the surface of soil in plastic containers, was determined after cultivation for 90 days outdoors. Its effect on plant growth and nutrient uptake was determined by growing Nostoc with Komatsuna (Brassica rapa var. peruviridis) on a soil-less culture in a plastic petri dish incubated inside a growth chamber for 14 days. Two colonies of Nostoc with different shapes were also examined for their tolerance to dryness and salinity. Nostoc application resulted in an increase in the organic carbon and nitrogen content of the surface soil and enhanced plant growth and plant iron uptake, but soil temperature and moisture were maintained. Spherical-shaped Nostoc showed a higher tolerance to dryness and salinity than irregular-shaped Nostoc, and the former was able to rectify problematic soil better than the latter. The results indicate that the application of Nostoc to soil has a potential for increasing soil organic matter and reclaiming degraded soil ecosystems. Presented at the 6th Meeting of the Asian Pacific Society of Applied Phycology, Manila, Philippines.     

Assessment of the metal removal capability of two capsulated cyanobacteria, Cyanospira capsulata and Nostoc PCC7936

Two capsulated, exopolysaccharide-producing cyanobacteria, Cyanospira capsulata and Nostoc PCC7936, were tested with regard to their metal removal capability by using copper as model metal. The experiments, carried out with the sole cyanobacterial biomass suspended in distilled water and confined into small dialysis tubings, showed that C. capsulata biomass is characterized by the best efficiency in metal removal, with a q_max (maximum amount of copper removed per biomass unit) of 96 ± 2 mg Cu(II) removed per g of protein in comparison with the value of 79 ± 3 of Nostoc PCC7936 biomass. The experimental data obtained with both cyanobacterial biomass best fit the Langmuir sorption isotherm. The sorption of copper started from the first minutes of contact with the metal and attained the equilibrium state, when no more copper removal was evident, after 5 and 6 hours, for C. capsulata and Nostoc PCC7936, respectively. The best efficiency in Cu(II) removal was obtained at pH 6.1–6.2, while the presence of Mg²⁺ or Ca²⁺ reduced copper removal capability of both species to 60–70% of their q_max. The results showed that the biomass of C. capsulata and Nostoc PCC7936 possesses a high affinity and a high specific uptake for copper, comparable with the best performances shown by other microbial biomass, and suggest the possibility to use the capsulated trichomes of the two cyanobacteria for the bioremoval of heavy metals from polluted water bodies.     

Exopolysaccharide-producing cyanobacteria and their possible exploitation: A review

Since the early 1950s, more than one hundred cyanobacterial strains,belonging to twenty different genera, have been investigated with regard tothe production and the released exocellular polysaccharides (RPS) into theculture medium. The chemical and rheological properties show that suchpolysaccharides are complex anionic heteropolymers, in about 80% casescontaining six to ten different monosaccharides and in about 90% casescontaining one or more uronic acids; almost all have non-saccharidiccomponents, such as peptidic moieties, acetyl, pyruvyl and/or sulphategroups. Based on such ingredients, cyanobacterial RPSs show promise asthickening or suspending agents, emulsifying or cation-chelating compoundsand the residual capsulated cyanobacterial biomass, following RPSextraction, could be an effective cation-chelating material. Indeed, wheneleven unicellular and filamentous RPS-producing cyanobacteria, selectedon the basis of the anion density of their RPSs and on the abundance oftheir outermost investments, were screened for their ability to removeCu²⁺ from aqueous solutions, a quick and most effective heavy metaladsorption was observed for the unicellular Cyanothece CE 4 and thefilamentous Cyanospira capsulata. These results suggest the possibilityto accomplish, through the exploitation of RPS-producing cyanobacteria,a multiproduct strategy to procure a wide range of biopolymers suited tovarious industrial applications, in addition to the residual biomass effectivein the recovery of heavy metals from polluted waters.     

Carbohydrate synthesis by two Navicula strains isolated from benthic and pelagic mucilages in the Tyrrhenian Sea (Tuscan Archipelago)

Two exopolysaccharide (EPS)-producing strains of the diatom Navicula, were isolated from benthic and pelagic mucilaginous aggregates sampled in the Tyrrhenian Sea and cultured under laboratory conditions. The amount of carbohydrate formed over the growth period and on a per cell basis was quite similar. However, the benthic strain showed a preferential synthesis of the bound (i.e., cellular and capsular) carbohydrate fraction, whereas the pelagic strain preferentially synthesised soluble, polymeric carbohydrates. The polysaccharides released into the medium by the two strains showed the same qualitative monosaccharidic composition, being constituted by two acidic and six neutral sugars. It is suggested that the difference between the benthic and the pelagic strain in the synthesis of bound or soluble carbohydrates may be related to the different role of these compounds in the particular habitats of the strains.     

The role of extracellular polysaccharides produced by the terrestrial cyanobacterium Nostoc sp. strain HK-01 in NaCl tolerance

The terrestrial cyanobacterium Nostoc sp. HK-01 was more tolerant to NaCl stress than the aquatic cyanobacterium Anabaena sp. PCC 7120 (also called Nostoc sp. PCC 7120) which is similar to Nostoc sp. HK-01 in phylogeny. We determined the amount of extracellular polysaccharides (capsular and released polysaccharides) from the cells of both strains cultured with or without 200 mM NaCl. The amount of capsular polysaccharides from Nostoc HK-01 reached approximately 65% of the dry weight whereas that from Anabaena PCC 7120 only occupied approximately 18% of the dry weight under NaCl stress. Anabaena PCC 7120 grew well under NaCl stress when both polysaccharides from Nostoc HK-01 were added to the culture. However, Anabaena PCC 7120 barely grew under NaCl stress when both of its polysaccharides were added. Extracellular polysaccharides from Nostoc HK-01 contained abundant fucose and glucuronic acid in comparison with those from Anabaena PCC 7120. Under NaCl stress, the composition ratios of sugars in the extracellular polysaccharides from Anabaena PCC 7120 hardly changed in comparison with those in ordinary culture conditions. By contrast, the composition ratios of sugars in the extracellular polysaccharides from Nostoc HK-01 changed under NaCl stress. These results suggest that the effect of extracellular polysaccharides from Nostoc HK-01 on NaCl tolerance comes from the increased amount of capsular polysaccharides, the sugar composition, and the change of the sugar composition ratio under NaCl stress.     

Differentiated pellicle organization and lipopeptide production in standing culture of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strains

Pellicle formation and lipopeptide production was analysed in standing cultures of different Bacillus subtilis strains producing two or three families of lipopeptides. Despite its ability to produce surfactin, B. Subtilis ATCC 6633 was unable to form stable pellicle at air–water interface. For the ATTC 21332 and ATCC 9943 strains, it was shown for the first time that the lipopeptides were also produced in standing cultures at productivities similar or lower than those obtained when the culture medium is agitated. A differentiated behaviour was observed between these strains in repetitive batch cultures. B. subtilis 9943 formed a wrinkled, thinner and more resistant pellicle than B. subtilis 21332. The structure of the pellicle determined by electron microscopy observations showed that cells of B. subtilis 9943 formed microcolonies whereas those of B. subtilis 21332 rapidly died. Under these conditions, surfactin production by strain 21332 decreased after 2 days whereas it remained stable for B. subtilis 9943 during the 6 days of the cultures. These data indicate that cells of B. subtilis strains growing in pellicle can produce lipopeptides differently depending on their cellular organisation. M. Chollet-Imbert and F. Gancel have contributed equally to the scientific work.     

Effect of heavy metals on the glutathione status in different ectomycorrhizal <Emphasis Type="Italic">Paxillus involutus</Emphasis> strains

The glutathione (GSH) status and heavy metal tolerance were investigated in four Paxillus involutus strains isolated from different heavy-metal-polluted and non-polluted regions of Europe. The heavy metal burden in the habitats did not affect significantly either the heavy metal (Cr₂O₇²⁻, Cd²⁺, Hg²⁺, Pb²⁺, Zn²⁺, Cu²⁺) tolerance and accumulation or the GSH production of the strains tested. Exposures to heavy metals increased the intracellular GSH concentrations in 12 from 24 experimental arrangements (four strains exposed to six heavy metals) independently of the habitats of the strains. The importance of GSH in heavy metal tolerance (high MIC values, ability to accumulate heavy metals and to grow in the presence of heavy metals) was thus demonstrated in this ectomycorrhizal fungus.     

Bioactivity in free-living and symbiotic cyanobacteria of the genus Nostoc

Fifty cyanobacterial strains from different habitats(symbioses, soil, fresh and marine waters) belongingto the genus Nostoc were cultured and tested forbioactivity. Thirty-seven strains were isolated in ourlaboratory, the remaining were supplied by officialculture collections. All the organisms were grownunder controlled laboratory conditions. The biomasseswere lyophilised and extracted with ethanol:water toobtain a hydrophilic extract and then withdichloromethane:isopropanol to obtain a lipophilicextract. Both crude extracts were tested forantifungal (against Penicillium expansum andRhizoctonia solani) and antibacterial activity(against Agrobacterium vitis, Escherichiacoli and Staphylococcus epidermidis), and fortoxicity against Artemia salina nauplii.Twenty-four strains showed activity against at leastone of the target organisms. Bioactivity was equallydistributed between lipophilic and hydrophilicextracts, and was mostly directed against fungi (15strains) and Artemia nauplii (12 strains);antibacterial activity was less frequent (8 strains).The presence of bioactivity was independent of thestrain origin.     

The released polysaccharide of the cyanobacterium Aphanothece halophytica inhibits flocculation of the alga with ferric chloride

The effect of the released polysaccharide (RPS) of the cyanobacterium Aphanothece halophytica GR02 on the recovery of the alga by flocculation with ferric chloride was studied. With increasing RPS concentration in algal cultures from 0 to 68 mg L⁻¹ the flocculation efficiency at the same dosage of ferric chloride decreased, and higher dosages of ferric chloride were required to attain the same flocculation efficiency. It is demonstrated that RPS could form complexes with ferrum during flocculation. In conclusion, RPS of A. halophytica GR02 had a significant inhibitory effect on flocculation of the alga with ferric chloride. The inhibitory mechanism of A. halophytica GR02 RPS allows the RPS to compete for ferrum by forming complexes with ferrum, thus leading to the consumption of ferrum in ferric chloride.     

Exopolysaccharide production by filamentous fungi: the example of Botryosphaeria rhodina

One-hundred and five fungal strains, belonging to 46 different species, were screened for exopolysaccharide production. Phytopathogenicity and, in particular, inability to produce conidia, were physiological characteristics positively associated and correlated with the fungal ability to produce polysaccharides. Among the 29 positive strains, Botryosphaeria rhodina DABAC-P82 was the most interesting reaching, when grown on optimal nitrogen source and concentration (NaNO₃ and 2.0 g l⁻¹, respectively) and culture medium pH (3.7), 17.7 g l⁻¹ of exopolysaccharide production after only 24 h of fermentation; yield and productivity were 0.69 g g⁻¹ and 0.73 g l⁻¹ h⁻¹, respectively. The purified polysaccharide was characterised as a homopolysaccharide of glucose with a molecular weight of 4.875·10⁶ Da. Studies of structural analysis indicated the presence of β-1,3 and β-1,6 linkages; the EPS structure was very similar to that of scleroglucan. This revised version was published online in August 2006 with corrections to the Cover Date.     

The role of exopolymers in the bioleaching of a non-ferrous metal sulphide

Exocellular polysaccharides were extracted from Thiobacillus ferrooxidans cells grown in the presence of iron. Cells without these compounds could not adhere to covellite. The loss of the layer of exocellular polysaccharides also affected the direct mechanism of bioleaching of covellite in a negative way. This ability to attach to and leach covellite was restored within a few hours when exopolymeric material was produced again. The addition of exocellular compounds to cells stripped of exocellular polymers also restored their ability to the same level as that of untreated cells. Thiobacillus thiooxidans was not able to attach to and leach covellite even when exocellular compounds from Thiobacillus ferrooxidans were added. Received 2 June 1998/ Accepted in revised form 8 January 1999     

Exopolysaccharide production by a new Halomonas strain CRSS isolated from saline lake Cape Russell in Antarctica growing on complex and defined media

A haloalkalophilic Halomonas strain CRSS, isolated from salt sediments in Antarctica, produced exocellular polysaccharides (EPS) up to 2.9gg^-1 dry cells. Acetate was the most efficient carbon source for EPS production. The composition of media strongly affected the nature of the polymers; a mannan and a xylo-mannan, were obtained when cells were grown on complex media. Acetate was the most efficient carbon source for EPS production and in presence of this substrate, a new polysaccharide, a fructo-glucan, was produced. The EPS fraction was composed by glucose, fructose, glucosamine and galactosamine in relative proportions of 1:0.7:0.3:trace.Revisions requested; Revisions received 6 September 2004     

MUCILAGINOUS CAPSULE ADSORPTION AND INTRACELLULAR UPTAKE OF COPPER BY KIRCHNERIELLA APERTA (CHLOROCOCCALES)1

The purpose of the present investigation was to evaluate possible ecological and physiological functions of mucilaginous capsules produced by the freshwater algae Kirchneriella aperta Teiling (Chlorococcales) as related to copper ions. All experiments were performed using synthetic media under laboratory‐controlled conditions. Copper interactions were investigated by distinguishing between adsorption onto the mucilaginous material present at the surface of the cells, intracellular uptake, and differentiation between total dissolved copper and free copper ions in the culture medium. Kirchneriella aperta is sensitive to copper, as revealed by a 96‐h EC₅₀ value of 10 ^{? 9.22} M Cu^{2 +} . We demonstrated that the mucilaginous capsules were able to sequester copper ions from the medium through a passive mechanism, thus providing the cell with a mechanism able to postpone the toxic effects of copper. The organic material that diffuses into the test medium as well as the mucilaginous capsules produced by K. aperta both effectively complex copper; thus, toxicity must be related to free copper ions and not the total dissolved copper concentration in the medium.     

Fixation of [13N]N2 and transfer of fixed nitrogen in the Anthoceros-Nostoc symbiotic association

The initial product of fixation of [¹³N]N₂ by pure cultures of the reconstituted symbiotic association between Anthoceros punctatus L. and Nostoc sp. strain ac 7801 was ammonium; it accounted for 75% of the total radioactivity recovered in methanolic extracts after 0.5 min and 14% after 10 min of incubation. Glutamine and glutamate were the primary organic products synthesized from [¹³N]N₂ after incubation times of 0.5–10 min. The kinetics of labeling of these two amino acids were characteristic of a precursor (glutamine) and product (glutamate) relationship. Results of inhibition experiments with methionine sulfoximine (MSX) and diazo-oxonorleucine were also consistent with the assimilation of N₂-derived NH ₄ ⁺ by Anthoceros-Nostoc through the sequential activities of glutamine synthetase (EC 6.3.1.2) and glutamate synthase (EC 1.4.7.1), with little or no assimilation by glutamate dehydrogenase (EC 1.3.1.3). Isolated symbiotic Nostoc assimilated exogenous ¹³NH ₄ ⁺ into glutamine and glutamate and their formation was inhibited by MSX, indicating operation of the glutamine synthetase-glutamate synthase (GS-GOGAT) pathway: However, relative to free-living cultures, isolated symbiotic Nostoc assimilated 80% less exogenous ammonium into glutamine and glutamate, implying that symbiotic Nostoc could assimilate only a fraction of N₂-derived NH ₄ ⁺ . This implication was tested by using Anthoceros associations reconstituted with wild-type or MSX-resistant strains of Nostoc incubated with [¹³N]N₂ in the presence of MSX. The results of these experiments indicated that, in situ, symbiotic Nostoc assimilated about 10% of the N₂-derived NH ₄ ⁺ and that NH ₄ ⁺ was made available to Anthoceros tissue where it was apparently assimilated by the GS-GOGAT pathway. Since less than 1% of the fixed N₂ was lost to the suspension medium, it appears that transfer of NH ₄ ⁺ from symbiont to host tissue was very efficient in this extracellular symbiotic association.Abbreviations DON 6-diazo-5-oxo-l-norleucine - GDH glutamate dehydrogenase - GOGAT glutamate synthase - GS glutamine synthetase - MSX l-methionine-dl-sulfoximine     

Rivularia halophila sp. nov. (Nostocales,Cyanobacteria): the first species of Rivularia described with the modern polyphasic approach

ABSTRACT

Natural populations of a Rivularia-like cyanobacterium were collected from the carbonate deposits of the temporarily flooded littoral zone of a hypersaline, high elevation lake, The Laguna Negra, Andes, Argentina. Subsequently, the cyanobacterial strain PUNA-NP3, named after its origin (Puna Volcanic Plateau) was isolated from these Rivularia-like rounded, pillow-like, black microbial mats. None of the previously described species of the genus Rivularia occupy inland, hypersaline aquatic environments. After morphological examination of this strain, we found clear morphological autapomorphies, such as mucilaginous pads at the bases of the young trichomes, wide trichomes and filaments, and uniquely branched trichomes. Furthermore, based on results from 16S rRNA phylogeny and analysis of the 16S-23S ITS region, PUNA-NP3 was found to be an independent lineage of the evolutionary tree. Based on the combination of ecological, morphological and molecular evidence, we name strain PUNA-NP3 Rivularia halophila sp. nov. a new species under requirements of the International Code of Nomenclature for Algae, Fungi and Plants.     

Double Mutants of Cellulomonas biazotea for Production of Cellulases and Hemicellulases following Growth on Straw of a Perennial Grass

Summary Deoxyglucose-resistant mutants of Cellulomonas biazotea secreted elevated levels of cellulases and xylanases. The production of β-glucosidase in the constitutive mutant was increased 5-fold over its parent strain. This mutant showed an approximately 1.6-fold enhanced productivity of extracellular endo-glucanase following growth on Leptochloa fusca over the mutant parent. Extracellular production of xylanase, filter-paper cellulase (FPase) and endo-glucanase (CMCase) were also altered in the mutant. Maximum volumetric productivities for xylanase, β-xylosidase, FPase, β-glucosidase and endo-glucosidase were 451, 98, 80, 95, and 143 IU l⁻¹ h⁻¹ which were significantly more than their respective values from the parental strains. The enzyme preparation of the mutants exhibited improved saccharification of kallar grass straw.     

Extracellular polysaccharide synthesis by Nostoc strains as affected by N source and light intensity

A study on the effect of two of the main factors affecting energy flux in N(2)-fixing cyanobacteria, i.e. light intensity and availability of combined nitrogen, on the synthesis of soluble exopolysaccharides was carried out with three strains of the genus Nostoc (PCC 7413, PCC 7936, and PCC 8113) presenting different capsular polysaccharidic morphologies and released polysaccharide productions. Strains acclimated to diazotrophic and non-diazotrophic conditions were cultured at high and low light intensities in aerated batch cultures. High light intensities enhanced total carbohydrate synthesis in all the strains but growth measured as pigment and protein concentration, total and soluble carbohydrate concentrations presented a strain-dependent response to nitrate availability. When adequately acclimated to the presence of nitrate all the capsulated strains tested became non-capsulated, with no extracellular polysaccharide being produced. Carbon availability can be on the basis of the observed correlation between the synthesis of capsular polysaccharides and diazotrophy. The slime-forming strain Nostoc PCC 7413 was the only one releasing polysaccharides into the surrounding medium under both, diazotrophic and non-diazotrophic conditions, with the highest values being obtained in the presence of nitrate. This strain presented the highest total carbohydrate (3.5 gl(-1)), soluble carbohydrate (1.8 gl(-1)) concentrations and viscosity values of all the tested strains. Different mechanisms of nitrogen-control of the synthesis of exocellular polysaccharides are reported for each strain, which results in the requirement of a species-specific optimisation of the cultivation conditions for the development of an efficient technology for the production of cyanobacterial exopolysaccharides.