Immobilization of Anabaena azollae from Azolla filiculoides in polyvinyl foam for ammonia production in a photobioreactor system

Anabaena azollae (AS-DS), isolated from Azolla filiculoides and grown in nitrogen-free medium, was immobilized in 5-mm-cube polyvinyl foam pieces and incorporated into a photobioreactor system for the production of NH₃. NH₃ was produced continuously and in significant amounts. Benlate (methyl-1-butyl-carbamoyl)-2-benzimidazole carbamate at 5 ppm and l-methionine-d,l-sulphoximine at 50 m stimulated NH₃ production continuously for a period of 1 week.S. Kannaiyan is with the Biotechnology Unit, Department of Agricultural Microbiology, Tamilnadu Agricultural University, Coimbatore-641003, Tamilnadu, India; K.K. Rao and D.O. Hall are with the Division of Life Sciences, King's College London, Campden Hill Road, London W8 7AH, UK.     

The ultrastructure of Anabaena azollae in Azolla pinnata

The water fern Azolla pinnata R. Br. was collected in Northwestern India and its structure was examined using scanning and transmission electron microscopy. Emphasis was given to the symbiotic cyanobacterium. Anabaena azollae , and to its relationship to the hairs of the leaf cavities. The cyanobacterial filaments are loosely arranged and often adhere to the protruding hairs and the folded cell walls of the cavities. The vegetative cells show a typical bilayered cell wall. Thylakoids are few and evenly dispersed in mature vegetative cells and appear to lack phycobilisomes. Clusters of polyglucoside granules are distinguished between the thylakoids. Thylakoid membranes are often seen forming whirls and lattices. Polyhedral bodies (carboxysomes) appear particularly frequently in younger cells and in the proximity of polyphosphate bodies. Presence of structured granules, often positioned at both sides of the cross-wall of neighbouring vegetative cells, suggest a positive nitrogen balance. A high frequency of heterocysts is noted, while spores are not observed.
The outer cell wall of the unbranched, mostly two-celled, hairs shows frequent invaginations. The cytoplasma of the mature hair contains numerous organelles, and is penetrated by an electron transparent network with blebs and vesieles appearing. The exchange of metabolites between the symbiotic partners is discussed in relation to the structures noted.     

Antigenic similarity among Anabaena azollae separated from different species of Azolla

Direct fluorescent antibody (FA) reaction results of 5 FAs against symbiotic Anabaena azollae indicated that all the A. azollae freshly separated from 32 specimens of Azolla collected worldwide (belonging to 6 different species) shared identical and highly specific antigens. None of these FAs exhibited cross-reaction with any of the free-living blue-green algae tested. FA absorption results confirmed these results and also indicate the existence of cross-reactive antigens between Azolla leaves and the surfaces of A. azollae. Antibodies made against free-living A. azollae did not cross-react with any of the symbiotic A. azollae indicating either: (i) these isolates are not true isolates, or (ii) their antigenic properties were altered during isolation and culturing. Such possibilities and their implications are discussed.     

The Azolla, Anabaena azollae Relationship: I. Initial Characterization of the Association

Cultures of Azolla caroliniana Willd. free of the symbiotic blue-green alga, Anabaena azollae, were obtained by treatment of Azolla fronds with a regimen of antibiotics. These symbiontfree plants can be maintained only on medium containing a combined nitrogen source.     

Production of polysaccharides by Arthrobacter globiformis associated with Anabaena azollae in Azolla leaf cavity

Abstract The composition of the capsular polysaccharides (CPS) and exopolysaccharides (EPS) of three strains of Arthrobacter globiformis , isolated from the leaf cavities of Azolla caroliniana (strain B1), A. filiculoides (strains A3 and L1) and A. globiformis ATCC 8010 have been analysed by HPLC and enzymatic assays. Glucose and galactose were detected in the EPS of all the strains, while rhamnose was present only in the EPS of the strain L1 and uronic acids in B1 and ATCC 8010. Traces of fructose were detected by enzymatic assays in all the strains. The CPS contained glucose, galactose and rhamnose, while uronic acids were present only in strain B1. In all the strains the amount of EPS was higher than CPS. The reactivity to different dyes and lectins of the mucilagineous matrix of the algal packets extracted from the fern and of the bacterial mucilage were similar.     

Antigenic differences between Anabaena azollae fresh from the Azolla fern leaf cavity and free-living cyanobacteria

Fluorescent antibody staining indicated differences in surface antigenicity in Anabaena azollae cells fresh from the leaf cavities of the fern, Azolla caroliniana, and algae which were isolated and subcultured from this fern. Such results suggest that either changes in antigenicity occur in this phycobiont during culturing or that isolation selects for an antigenically different mutant strain capable of in vitro growth.Non-Standard Abbreviations FA fluorescent antibody staining - PBS phosphate buffered saline - W microwatt - Anti-F antiserum prepared against fresh cells - Anti-N antiserum prepared against Newton's culture - FTTC fluorescein isothiocyanate To whom offprint requests should be sent     

Metal removal by immobilised and non-immobilised Azolla filiculoides

Milled-sieved and epichlorhydrin-immobilised Azolla biosorbed ca. 363 and 320 mol Cu²⁺ g^–1 from a 100 mg l^–1 solution. Efficiency of Cu²⁺ removal by columns was in the order epichlorohydrin-immobilised Azolla>milled-sieved Azolla>untreated Azolla. The 2.5 g epichlorohydrin-immobilised Azolla column demonstrated complete metal sequestration from ca. 12 l of influent 5 mg Cu²⁺ l^–1 and was still at less than 75% saturation even after ca. 22 l had passed through the column. EDTA effectively desorbed Cu²⁺ with a ca. 55-fold decrease in volume.     

Growth and survival of Azolla filiculoides in Britain. II. Sexual reproduction

Sporulation in the floating fern Azolla filiculoides Lam. is both frequent and widespread in Britain and might therefore play a greater part in the population dynamics of the species than has been suggested by earlier reports. In laboratory experiments, increasing plant density and/or phosphorus supply resulted in increased sporulation. It was estimated that a thick mat of 8 kg m² fresh biomass can produce 380000 microsporocarps and 85000 megasporocarps per m².
Light and temperatures >10°C were necessary for sporocarp germination. Sporocarps could survive exposure to both low temperatures (5°C for at least 3 months) and sub-zero temperatures (−10°C for at least 18 d). Sporocarps were found to survive storage in water for 3 yr and to germinate from mud samples collected in the field. In laboratory culture, sporeling growth and survival were optimal at 15°C.
There is some evidence that A. filiculoides might have adapted to the British climate since its introduction.     

Growth and survival of Azolla filiculoides in Britain I. Vegetative production

Azolla filiculoides Lam. causes serious weed problems in Britain, but its long-term survival might be limited by winter death. The aim of this study was to establish the low temperature responses and limitations of A. filiculoides sporophytes.
In the laboratory, normal vegetative growth was shown to continue at 5°C. Reddening of plants was a response to low temperature and high light conditions which could be prevented by shading. Adult plants died after short (18 h) exposure to −4°C but survived sub-zero temperatures >−4°C. Evidence was found of seasonal changes in chill tolerance, but not in freeze tolerance.
In outdoor culture, plants survived encasement in ice and air temperatures to −5°C. Additional evidence suggested that natural populations can readily survive air temperatures much lower than this. Microclimatic effects are likely to be responsible for this discrepancy between laboratory and outdoor culture results.
Three phenotyes were identified; survival, colonizing and mat forms.     

Isolation of Hair Cells from Azolla filiculoides var. japonica Leaves

A procedure has been developed to isolate hair cells from Anabaenacontaining packets of the aquatic fern Azolla. Unbroken algalpackets, isolated by enzyme treatment and flotation on 12.5%Percoll solution, contained hair cells, Anabaena filaments andthe envelope membrane. When the packet suspension was gentlypipetted, hair cells attached to the envelope membrane becamedetached from the Anabaena filaments. Hair cells were separatedby flotation on 34% Percoll solution and further purified bysifting through nylon mesh. Both branched and unbranched cellswere isolated and about 70% remained unstained by 0.1% trypanblue. Electron microscopic observation showed that these cellswere protoplasts enclosed by a thin envelope and had well preservedinternal structures. The activities of ammonia-assimilatingenzymes in the hair cells were much higher than those in Azollaleaves, while the activities in the endophytes were repressedto very low levels. These results suggest that hair cells playan important role in the assimilation of the nitrogen whichthe endophytes fix and release into the cavity. (Received March 24, 1986; Accepted June 28, 1986)     

Response of growth and antioxidant enzymes in Azolla plants (Azolla pinnata and Azolla filiculoides) exposed to UV-B

Effect of ultravilolet-B (0.4 Wm(-2)) irradiation on growth, flavonoid content, lipid peroxidation, proline accumulation and activities of superoxide dismutase and peroxidase was comparatively analysed in Azolla pinnata and Azolla filiculoides. Growth measured as increment in dry weight reduced considerably due to all UV-B treatments. However, the reduction was found to be severe in A. filiculoides as compared to A. pinnata. The level of UV-absorbing compound flavonoids increased significantly in A. pinnata plants whereas only a slight increase in the flavonoid content was observed in A. filiculoides. UV-B exposure led to enhanced production of malondialdehyde (MDA) and electrolyte leakage in A. filiculoides than A. pinnata. Proline accumulation also showed a similar trend. Marked differences in the activity of antioxidant enzymes such as superoxide dismutase (SOD) and peroxidase (POD) was noticed in both the plants exposed to UV-B. Our comparative studies indicate A. pinnata to be better tolerant to UV-B as compared with A. filiculoides which appears to be sensitive.     

Genetic diversity and phylogeny analysis of Anabaena azollae based on RFLPs detected in Azolla-Anabaena azollae DNA complexes using nif gene probes

The cyanobacterium Anabaena has both symbiotic and free-living forms. The genetic diversity of Anabaena strains symbiotically associated with the aquatic fern Azolla and the evolutionary relationships among these symbionts were evaluated by means of RFLP (restriction fragment length polymorphism) experiments. Three DNA fragments corresponding to nif genes were cloned from the free-living cyanobacterium Anabaena PCC 7120 and used as probes. A mixture of Azolla, Anabaena and bacterial DNA was extracted from Azolla fronds and digested with two restriction enzymes. Single-copy RFLP signals were detected with two of the probes in all Azolla Anabaena examined. Multiple-copy RFLP signals were obtained from the third probe which corresponded to a part of the nif N gene. A total of 46 probe/enzyme combinations were scored as present or absent and used to calculate pairwise Nei's genetic distances among symbiotic Anaebaena strains. Phylogenetic trees summarizing phenetic and cladistic relationships among strains were generated according to three different evolutionary scenarios: parsimony, UPGMA and neighbour joining. All trees revealed identical phylogenetic relationships. Principal component analysis was also used to evaluate genetic similarities and revealed three groups: group one contains the cyanobacteria associated with plants from the Azolla section, group two contains those associated with plants from the pinnata species and group three contains those associated with plants from the nilotica species. The same groups had already been identified earlier in a random amplified polymorphic DNA (RAPD) analysis of Azolla-Anbaena DNA complexes, suggesting that the present Azolla taxonomy should be revised. We now suggest a taxonomy of Anabaena azollae that is parallel to such a revised Azolla taxonomy. An Azolla chloroplast DNA sequence derived from Oryza sativa was also used as an RFLP probe on Azolla DNA to confirm the presence of plant DNA in the total genomic DNA extracted from ferns with or without the symbiont. Our results also suggest that total DNA extracted from the Azolla-Anabaena complexes includes both plant and symbiont DNA and can be used equally well for RFLP analysis of host plant or symbiotic cyanobacteria.     

Characterization of akinete differentiation in the cyanobacterium Anabaena azollae

Differentiation of akinetes was investigated in the filamentous cyanobacterium Anabaena azollae Stras. In this organism all pre-existing vegetative cells are capable of developing into akinetes. Standard sporulation medium (SSM) was used to synchronously induce the formation of akinetes, while cultures in Allen and Arnon (AA/8) medium were used as controls.This paper describes the changes in photosynthetic pigments and total soluble proteins in these cultures over a 25-day period encompassing akinete differentiation. Heterocyst frequencies and nitrogenase activity were also monitored during the same period in both media. SDS-PAGE results indicated that specific proteins were synthesized in a manner correlated with akinete differentiation. The results demonstrate that in cultures undergoing akinete development, some of the photosynthetic pigments are maintained, nitrogen-fixation and heterocyst differentiation are suppressed, and the cells synthesize a variety of specific proteins.     

Variation revealed by RAPD‐PCR profiles of microsymbiont Anabaena azollae strains from four N2 fixing Azolla cultures

Nitrogen fixing Anabaena azollae strains isolated from four different Azolla cultures were characterized based on their total protein profile and RAPD profile to study the existing variation among them. As expected, the isolates showed almost similar protein banding patterns, but exhibited differences in 40–70 KDa protein subunits. Polymerase chain reaction of the DNA of the isolates, using four different primers, amplified specific sequences of DNA and showed clear polymorphism among the isolates. The RAPD profile generated the fingerprinting pattern characteristic of each strain based on the sequence of the primers used. Common band sharing observed between the strains A. azollae‐RS‐KK‐SK‐AM and A. azollae‐RS‐KK‐SK‐RP probably represents maternal inheritance of DNA to the progeny. The polymorphic bands were generated specifically for the isolates A. azollae‐RS‐KK‐SK‐RP and A. azollae‐RS‐KK‐SK‐AM with primers numbered 2 and 4, respectively, which could be developed as possible markers for these isolates.     

Performance of Dead Azolla filiculoides Biomass in Biosorption of Au from Wastewater

Dried milled biomass of Azolla filiculoides removed up to 98.2% of gold from wastewater from a gold plating factory containing 5 mg gold/l in solution in batch biosorption. The gold uptake capacity of the biomass was 98 mg/g. Whole dried biomass used in a continuous flow column removed up to 100% of gold from diluted wastewater. A similar column was linked to a sulphide precipitation process to provide a two-step system which was able to remove 98% of gold from undiluted wastewater containing 41 mg Au/l. The lifetime of the column was five days.     

The effect of immobilization on carbon flow through Anabaena variabilis and A. azollae

Immobilization in polyvinyl foam resulted in an increased carbon fixation and release of fixed carbon in Anabaena variabilis while in A. azollae both processes decreased. This revised version was published online in June 2006 with corrections to the Cover Date.     

Megaspore germination, embryo development and maintenance of the symbiotic association in Azolla filiculoides Lam.

Megaspore germination and embryo development in Azolla filiculoides was examined using SEM and thin–sectioning. Within the released megaspore apparatus, resting cells of the endosymbiont Anabaena azollae Stras. arc located distally to the outside of the mcgasporangial wall and adhering to the inside of the megasporocarp wall. Growth of the female gametophyte displaces the floats pushing this part of the wall (the indusial cap) upwards, so providing access to the archegonia for the multifiagellalc spermalozoids. Embryo development and its inoculation with Anabaena involves a subtly–timed sequence of events resulting in the perpetuation of the symbiosis. Growth of the lunnel–shaped cotyledon leaf ruptures the mcgasporangial wall to provide access and a channelled route between the Anabaena and embryo shoot apex; subsequent leaf development severely restricts such access. During this process, the Anabaena is dislodged by the cotyledon leaf and growth of the first leaf traps the now actively–dividing Anabaena colonv; this becomes established around subapical trichomes from where filaments become incorporated into the cavities of developing leaves. The voung sporophyte rises vertically to the water surface as a result of gas accumulation in intercellular spaces; at no stage do floats endow buoyancy.     

The effect of immobilization on carbon flow through Anabaena variabilis and A. azollae

Immobilization in polyvinyl foam resulted in an increased carbon fixation and release of fixed carbon in Anabaena variabilis while in A. azollae both processes decreased.