Frankia Growth Research Articles

Neighbours in nodules: the interactions between Frankia sp. ACN10a and non-Frankia nodular endophytes of alder.

In the present study, we report the in vitro interactions between Frankia sp. ACN10a and non-Frankia nodular endophytes (NFNE) isolated from alder. The supernatant of NFNE grown in nitrogen-replete medium had neutral or negative effects on Frankia growth; none had a stimulatory effect. Inhibitory effects were observed for supernatants of some NFNE, notably Micromonospora, Pseudomonas, Serratia and Stenotrophomonas isolates. However, some NFNE-Frankia coculture supernatants could stimulate Frankia growth when used as a culture medium supplement. This was observed for supernatants of Frankia cocultured with Microvirga and Streptomyces isolates. In nitrogen-limited conditions, cocultures of Frankia with some NFNE, including some rhizobia and Cytobacillus, resulted in higher total biomass than Frankia-only cultures, suggesting cooperation, while other NFNE were strongly antagonistic. Microscopic observation of cocultures also revealed compromised Frankia membrane integrity, and some differentiation into stress resistance-associated morphotypes such as sporangia and reproductive torulose hyphae (RTH). Furthermore, the coculture of Frankia with Serratia sp. isolates resulted in higher concentrations of the auxinic plant hormone indole-3-acetic acid (IAA) and related indolic compounds in the culture supernatant. This study sheds new light on the breadth of microbial interactions that occur amongst bacteria that inhabit the understudied ecological niche of the alder nodule.

Hydroxypyridinones in nitrogen-fixing bacterial cultures: a metal buffer for molybdenum and simulation of natural conditions.

Organic matter regulates the availability of important trace elements in aquatic and terrestrial ecosystems by acting as a source and container for microbes. To overcome the limitation of trace elements, nitrogen-fixing bacteria, e.g. release low-molecular-weight chelators (metallophores), which scavenge the essential cofactors of the nitrogenase, iron, and molybdenum (Mo), via complexation and subsequent uptake. The formation of metallophores is triggered by limiting conditions, which must be replicated in the laboratory in order to study metallophores as a mediator in metal cycling. While ethylenediaminetetraacetic acid (EDTA)-based buffer systems for metal cations are well established, there is limited knowledge regarding the buffering of oxoanions such as molybdate in a bacterial growth medium. To mimic the availability of molybdenum in nature under laboratory conditions, this study created a Mo-buffer system for bacterial growth media of the model organisms Azotobacter vinelandii and Frankia sp. CH37. We investigated selected hydroxypyridinones (HPs) as potential molybdenum-chelating agents, determining the amount required for efficient molybdenum complexation by calculating speciation plots of the various candidate complexes in artificial growth media at various pH values. The Mo-maltol system was identified as an ideal, nontoxic molybdenum-buffer system. In the presence of the Mo-maltol system, the growth of Frankia sp. was limited under diazotrophic conditions, whereas A. vinelandii could acquire molybdenum through the release of protochelin and subsequent molybdenum uptake. The study paves the way for unravelling molybdenum recruitment and homeostasis under limiting conditions in bacteria.

Bioremediation of noxious metals from e-waste printed circuit boards by Frankia

The environmental noxious e-waste was collected and physicochemical characterized by Scanning electron microscopy (SEM) along with energy dispersive X-ray spectroscopy (EDX), Atomic absorption spectrometry (AAS), and X-ray diffraction analysis (XRD) exploration to understand the presence of toxic metals like Hg, Cd, Pd, Si, Ru. Therefore, the finding provides vital knowledge about the impact of toxic metals from e-waste printed circuit boards as contaminants in the environment and its impact on humans. The Frankia sp. DDNSF-03 and Frankia casuarinae DDNSF-04 were isolated and identified, further utilized for removal of e-waste toxic metals by one and two steps bioremediation experiments executed with various e-waste concentrations. The two-step bioremediation experiment is efficient in the expression of toxic metals that were removed at a lesser concentration of e-waste. Consequently, the presence of organic acids in the Frankia primary metabolites was confirmed by FT-IR analysis besides decreasing the pH level in the Frankia growth medium. The positive control Frankia and negative control e-waste were maintained throughout the bioremediation experiments. The initial Hg 4.3, Cd 8.3, Pd 4.6 (ppm) in the e-waste and final treated with Frankia sp. DDNSF-03 Hg 0.09, Cd 5.09, Pb 0.49 (ppm), and Frankia casuarinae DDNSF-04 Hg 2.15, Cd 5.6, Pb 2.82 (ppm) concentration of toxic metals was quantified by AAS spectrum analysis. The toxic metals mercury and lead were significantly mineralized by Frankia sp. when compare the Frankia casuarinae. The above finding was confirmed the manifestation of morphological changes by an accumulation of e-waste in Frankia hyphae using SEM analysis and obtain the qualitative of toxic metals parallel peaks in EDX analysis.

Effect of neem (Azadiracta indica) aqueous extract on Casuarina equisetifolia seed germination, growth of Frankia and some rhizospheric microorganisms.

Aqueous neem (Azadiracta indica) leaf extract was tested for its effect on Casuarina equisetifolia seed germination capacity, growth of Frankia and on some dominant rhizosphere microorganisms under casuarinas grown in soil contaminated with sewage water. Analysiswas carried out to determine the main micro- and macroelement content of different extract amounts. The addition of extract amount between 10- 20 µl per 10 ml medium (1- 2 µl ml-1) selectively stimulated Frankia growth and inhibited other non- beneficial bacteria and fungi in the rhizosphere such as Bacillus subtilis, Drechslera sp and Curvularia lunata. Slight increase was observed in Casuarina seed germination capacity with 1- 2 Μ ml-1 of the extract, but with no significant differences, indicating at least no negative effect of the extract on seed germination. According to the results and as the roots and leaves of both Casuarina and neem release their active components in soil, and according to previous recommendations, we suggest a significant synergistic effect between Casuarina, rhizosphere microorganisms and neem trees for the benefits of Casuarina nodulation, growth performance and nitrogen fixation. Key words: Neem, Casuarina, aqueous extract, Frankia, rhizospheric microorganisms.

Are Northwestern Patagonian “mallín” wetland meadows reservoirs of Ochetophila trinervis infective Frankia?

“Mallin” (plural mallines) is a particular kind of wetland occurring in Patagonian steppe and forests. In Northwest Patagonia, mallines are humid meadows with high net primary production. It was previously found that a mallin soil in the steppe devoid of actinorhizal plants had a higher Frankia nodulation capacity in Ochetophila trinervis (sin. Discaria trinervis) than other soils in the region. Under the hypothesis that mallin wetland meadows are reservoir of infective Frankia, we studied the Frankia nodulation capacity in O. trinervis of 12 mallin and their neighbouring steppe soils, by using plant bioassays. A qualitative plant bioassay showed that infective Frankia was present in most soils. The number of nodules per plant in seedlings inoculated with mallin soils was negatively correlated with soil water content while the opposite was true for plants inoculated with soils from neighbouring steppe. A quantitative bioassay was performed with eight representative soils, selected according to the number of nodules per plant produced in the qualitative assay and to the presence or not of different actinorhizal plants at the sites. Frankia nodulation units per cm3 of soil (NU) in mallin soils were higher than those in steppe. Water and organic matter content of soils were correlated with the higher nodulation capacity of mallines, which may account for the saprotrophic growth of Frankia in soils. The symbiosis was effective in plants inoculated with all soil samples. These results suggest that Northwestern Patagonian mallin wetland meadows are reservoirs of infective and effective Frankia propagules in O. trinervis.

Casuarina cunninghamiana tissue extracts stimulate the growth of Frankia and differentially alter the growth of other soil microorganisms.

Aqueous extracts of host plant Casuarina cunninghamiana tissue altered the in vitro growth of its diazotrophic microsymbiont Frankia and a selection of other soil microorganisms. The growth of actinomycetous Frankia strains, 55005. AvcI1, CesI5, CjI82 001, and Cj was stimulated by aqueous extracts of C. cunninghamiana tissue. Green cladodes (photosynthetic branches), unsuberized roots, and suberized roots were more stimulatory than dry cladodes and seed tissue. Aqueous extracts of green cladodes of C. cunninghamiana most stimulated the growth of Casuarina-derived Frankia strains CjI82 001 and 55005. The growth of isolates of soil bacteria Bradyrhizobium japonicum, Arthrobacter globiformis and Bacillus subtillis and of the soil fungi Penicillium oxalicum and Arthroderma cookiellum was either inhibited or not affected by cladode extracts. Cladode extracts stimulated the growth of the actinomycete Streptomyces albus and the fungus Rhizopus homothallicus. The magnitude (as great as 100%) of the increase in growth caused by tissue extracts for the Casuarina-derived Frankia strains relative to other soil microbes suggests a host-specific enhancement of the microsymbiont.

Improving dispersed growth of Frankia using Carbopol

Improving dispersed growth of Frankia using Carbopol

Boron requirement in the Discaria trinervis (Rhamnaceae) and Frankia symbiotic relationship. Its essentiality for Frankia BCU110501 growth and nitrogen fixation.

The essentiality of boron (B) for nitrogen fixation in heterocystous cyanobacteria and rhizobial symbioses has been widely established. However, nothing is known about the possible involvement of the micronutrient in actinorhizal symbioses. Therefore, the effect of boron (B) deficiency on the establishment of the Discaria trinervis-Frankia BCU110501 symbiosis was investigated. Nodulation was diminished in B-deficient D. trinervis or in plants inoculated with Frankia grown in the absence of B. These poorly nodulated plants showed a reduction of shoot and root weight and small size. Because depletion of the micronutrient during growth of the actinomycete altered the infection capacity of Frankia, we also studied growth, structure and nitrogen fixation of free-living Frankia BCU110501. Growth was delayed in B-deficient BAP media (+N cultures), and completely inhibited in B-deprived N-free BAP media (-N cultures), suggesting that B is required to enhance growth of Frankia and essential for the development of nitrogen fixing activity. Ultrastructural study of B-deficient cells showed an alteration of filament walls both in +N and especially in -N cultures, indicating a possible role of the microelement in the maintenance of these structures. Moreover, the stability of vesicle envelopes was impaired in the absence of B and, hence, nitrogenase occurrence and nitrogen fixation were totally absent. The results show that B is required for both partners to establish an effective symbiosis.

Effect of Al, Co, and Pb ions on growth of Frankia spp. in a mineral medium.

Growth of three Frankia strains associated with roots of Casuarina tree, treated with different concentrations of metal ions, was measured as total protein content. One strain was highly resistant to different aluminum ion concentrations up to 10 mmol/L. The other two strains were more sensitive to the higher aluminum concentrations (1.25-10 mmol/L). Growth inhibition by cobalt and lead concentrations varied, depending on the tested strain. Stimulation occurred only at cobalt concentrations of 0.33 and 0.65 mmol/L for one strain.

Stimulatory effects of aluminum on in vitro growth ofFrankia

To investigate the effects of aluminum (Al) on in vitro growth (total protein content) of Frankia, seven isolates were incubated for 25 days at five nominal Al concentrations (0-500 µM) at pH 4.8. The concentrations of monomeric Al ranged between 25.6 and 106.7 µM. The experimental medium was devoid of inorganic phosphate and Fe-EDTA to avoid Al precipitation. There was an Al-free control at pH 6.4 to assess the effects of low pH alone on Frankia growth. Growth estimated as total protein of all strains was notably increased at pH 6.4, but only Frankia CcI3 was able to grow in the Al-free medium at pH 4.8. Nominal Al concentrations from 125 to 500 µM enhanced the growth of all Frankia isolates. The Al response was strain dependent, but all strains were stimulated at the 500 µM (107 µM monomeric) Al concentration. The final pH of media decreased as Al concentrations increased. At the end of the experiment, the presence of citric, oxalic, malic, and lactic acids as possible Al binding agents in media at 500 µM Al was assayed by enzymatic techniques. However, none of them could be detected. These results suggest that the survival and growth of Frankia strains in acid soils that are characteristically high in Al are more likely than previously estimated from in vitro growth assays.Key words: acid soils, actinorhizal plants, aluminum, Frankia.

In vitro culture of arbuscular mycorrhizal fungus and Frankia for inoculation of micropropagated Casuarina equisetifolia L.

Micropropagated, rooted, and calli explants of Casuarina equisetifolia L. were inoculated with Frankia UGL 020605S and the arbuscular mycorrhizal fungus (AMF) Glomus mosseae, in single and dual co-culture, in vitro. Different micropropagation media formulations were evaluated for their capacity to stimulate germination of G. mosseae spores and growth of Frankia. Murashige and Skoog basal nutrient (half strength) medium, supplemented with 6-benzylaminopurine (BAP), 2,4-dichlorophenoxyacetic acid (2,4-D), and pyruvate was selected for the in vitro co-culture of C. equisetifolia callus explants, G. mosseae, and Frankia. This medium (M4) supported 70% AMF spore germination with 44 and 34% of the germinating spores producing single and branched hyphal strands, respectively. Hoaglands (quarter strength, modified by Hoaglands and Arnon (1950)) nutrient medium (M5) with no supplements was selected for the in vitro co-culture of rooted C. equisetifolia explants, G. mosseae, and Frankia and supported 57% AMF spore germination with 29 and 40% of the germinating spores producing single and branched hyphal strands, respectively. Both media supported significant growth of Frankia. In both cases agar was substituted with Terragreen(r). AMF appressoria and intercellular hyphae were observed in rooted C. equisetifolia at 28 days; arbuscule formation occurred at 56 days postinoculation. Frankia infection was evident after 28 days. This was observed in both dual and single in vitro co-cultures. No specific immunofluorescent or immunogold reactions to monoclonal antibodies (mABs) anti-Frankia < 8C5 > and anti-G. mosseae < F5G5 > were evident in C. equisetifolia callus explants.Key words: arbuscular mycorrhizal fungi (AMF), Frankia, Casuarina, micropropagation, immunofluorescent labelling.

Actinorhizal symbioses and their N2 fixation.

More than 200 angiosperms, distributed in 25 genera, develop root nodule symbioses (actinorhizas) with soil bacteria of the actinomycetous genus Frankia. Although most soils studied contain infective Frankia, cultured strains are available only after isolation from root nodules. Frankia infects roots via root hairs in some hosts or via intercellular penetration in others. The nodule originates in the pericycle. The number of nodules in Alnus is determined by the plant in an autoregulated process that, in turn, is modulated by nutrients such as nitrogen and phosphate. Except in the genera Allocausarina and Casuarina, Frankia in nodules develops so-called vesicles where nitrogenase is localized. Sporulation of Frankia occurs in some symbioses. As a group, actinorhizal plants show a large range of anatomical and biochemical adaptations in order to balance the oxygen tension near nitrogenase. In symbioses with well aerated nodule tissue like Alnus, the vesicles have a multilayered envelope composed mainly of lipids, bacterio-hopanetetrol and their derivatives. This envelope is assumed to retard the diffusion of oxygen into the nitrogenase-containing vesicle. In symbioses like Casuarina, the infected plant cells themselves, rather than Frankia, appear to retard oxygen diffusion, and high concentrations of haemoglobin indicate an infected region with a low oxygen tension. At least in Alnus spp., ammonia resulting from N2 fixation is assimilated by glutamine synthetase in the plant. The carbon compound(s) used by Frankia in nodules is not yet known. Nitrogenase activity decreases in response to a number of environmental factors but recovers upon return to normal conditions. This dynamism in nitrogenase activity is often explained by loss and recovery of active nitrogenase and has been traced to loss and recovery of the nitrogenase proteins themselves. Recovery is partly due to growth of Frankia and to development of new vesicles in the Alnus nodules. In the field, varying conditions continuously affect the plants and the measured rate of N2 fixation is a result not only of the conditions prevailing at the moment but also of the conditions experienced over preceding days. N2 fixed by actinorhizal plants is substantial and actinorhizal plants have great potential in soil reclamation and in various types of forestry. Several species are also useful in horticulture. CONTENTS Summary 375 I. Introduction 376 II. The partners of actinorhizal symbioses 377 III. Root nodules 380 IV. Nitrogen fixation and related processes 385 V. Environmental effects on nitrogen fixation 389 VI. Ecological role 397 VII. Concluding remarks 398 Acknowledgements 398 References 398.

Specific long-chain fatty acids promote optimal growth of Frankia: accumulation and intracellular distribution of palmitic and propionic acid

Frankia isolates from nodules of the genera Casuarina (BR, S21, Thr), Allocasuarina (Allo2), and Gymnostoma (G80) were found to grow exponentially with high biomass yield and minimal sporangia formation in stirred propionate mineral medium when supplemented either with 2.4 μM palmitic acid (C16:0), pentadecanoic (C15:0), heptadecanoic (C17:0), or linoleic (C18:2, cis 9, 12) fatty acids. Strains also grew with lauric (C12:0) or myristic (C14:0) acids, but gave lower biomass yield. Stearic acid (C18:0) produced a good biomass yield, but cultures slowly accumulated sporangia; oleic acid (C18:1, cis-9) was detrimental to growth. Caprylic (C8:0) or capric (C10:0) acids proved to be prejudicial for long-term storage of Frankia strains. In experiments using labeled 1,2-dipalmitoyl phosphatidylcholine and palmitic acid, radioactivity bound rapidly to the insoluble, but solvent-extractable fraction of Frankia cells. In contrast, label from propionic acid accumulated in the cytosolic fraction. Therefore, the beneficial effect of some specific phospatidylcholines or free fatty acids on Frankia growth appears to result from their utilization as building blocks for the membrane, suggesting that membrane biosynthesis may be the limiting step for Frankia growth in unamended propionate mineral medium.

Les mycorhizes des plantes actinorhiziennes

Summary As pioneer species for land revegetation, actinorhizal plants are predisposed to form with mycorrhizal fungi symbiotic associations that are as essential for plant growth as the associations they form with Frankia. As expected, mycorrhizas stimulate the development and the N2-fixing activity of the actinorhizal symbiosis by improving mineral nutrition of the host plant. In addition to mycorrhizas, actinorhizal plants are also able to form an unique type of roots called « proteoid roots » or « cluster roots » in response to the detrimental effects of nutrient deficiencies in soil. As an alternative of mycorrhizas, cluster roots are specialized roots capable of assisting plant growth in marginal, nutrient deficient soils. There is no competition between Frankia and mycorrhizal fungi for infection sites but extensive mycorrhizas can affect Frankia growth and the subsequent development of young lobes of actinorhizal nodules. By contrast, the presence of Frankia in planta has no incidence on mycorrhizal infection processus. With reference to Alnus and Allocasuarina the possible implications of Frankia and mycorrhizal fungi on the mechanisms of recognition and chemical signalisation between the host plant and these microsymbionts are discussed. Strikingly, it should be noticed that Frankia and arbuscular endomy- corrhizal fungi exhibit a number of similarities that could be taken into account for developing future research.

Effects of extracts of Alnus glutinosa seeds on growth of Frankia strain ArI3 under static and fermentor culture conditions

In the present study, the effect of Alnus glutinosa seed extract on the in vitro growth of Frankia strain ArI3 was investigated under various culture conditions. Frankia filaments grown in static cultures showed a very slow growth with doubling time of 12 days. However, under the same growth conditions but with seed extract added to the medium, the doubling time was reduced to 6 days. A further reduction in doubling time (2.5 days) was obtained without seed extract when Frankia strain ArI3 was grown in a fermentor with continuous stirring. Under these culture conditions, seed extract did not influence the Frankia growth rate. In all our studies, Na-propionate was used as the only carbon source. Gradual addition of Na-propionate to Frankia cultures in the fermentor sustained exponential growth of the filaments over a prolonged period. The complete consumption of Na-propionate was the limiting growth factor under fermentor growth conditions. The mechanism of growth stimulation by Alnus glutinosa seed extract under growth limiting conditions in static cultures and its possible ecological significance is discussed.

The effect of phosphorus on nodule formation and function in the Casuarina-Frankia symbiosis

A study was conducted to investigate the effects of phosphorus on nodule formation and function in the Casuarina-Frankia symbiosis. The effects of P on growth and survival of Frankia in the rhizosphere was assessed by examing Frankia growth and survival in flasks of basal nutrient solution. There was no growth in the nutrient solution during the experimental period. However, the viability of Frankia in the nutrient solution without P supply was half that of the initial level, whereas, with P supply, there was only a minor decline during the first week. In a growth pouch experiment, supplying P increased plant and nodule growth, irrespective of P status of the inoculant Frankia culture. There were no effects of P status on any growth or nodulation parameters measured when the inoculants had been standardized on the basis of viability. In a split root experiment, Frankia inoculation and application of P together or separately did not cause any significant difference. This suggests that growth and nodulation respond only to total P supply. Increasing P from 0.1 to 10 μM significantly increased plant growth but not N concentrations. Both nitrogen-fixation and nitrate supported growth were strongly increased as P increased from 0.1 to 1.0 μM. This study indicates that P deficiency limits the growth of host plants more severely than nitrogen fixation processes and P deficiency on nodulation and symbiotic nitrogen fixation in Casuarina cunninghamiana operated indirectly via reducing host plant growth.

1,2-dipalmitoyl phosphatidylcholine, 1,2-dipalmitoyl phosphatidic acid or 1,2-dipalmitoyl- sn-glycerol inhibit sporangia formation and promote exponential growth of various frankia isolates from the casuarinaceae family

Frankia strains from Casuarina (BR, S21, Thr), Allocasuarina (Allo2) and Gymnostoma (G80) genera were found to grow exponentially in stirred, propionate-containing BAP medium supplemented with 1.2 or 2.4 μ m synthetic 1,2-dipalmitoyl phosphatidylcholine, 1,2-dipalmitoyl phosphatidic acid or 1,2-dipalmitoyl- sn-glycerol. Phosphatidylcholines containing C15:0 or C17:0 acyl residues instead of C16:0 (palmitoyl) residues were equally beneficial but stearoyl (C18:0) residues favored the formation of small sporangia. Oleoyl residues (C18:1) appeared to be toxic as they produced a progressive post-exponential decrease in biomass. Our results suggest that phospholipids containing palmitoyl residues are part of the beneficial compounds present in the complex egg yolk phosphatidyl choline mixture which promote exponential growth of Frankia strains, inhibit sporangia formation and delay post-exponential biomass degradation. Phosphatidic acids do not appear to mediate the enhancing effect of phosphatidylcholine derivatives. The fact that 1,2-dipalmitoyl- sn-glycerol alone is beneficial, suggests that the phosphate and choline residues of the phosphatidylcholine molecule are not essential for balanced growth of Frankia under our conditions. Taken together our results suggest that a primary target of the beneficial compounds may be the plasma membrane of Frankia cells.

Inoculant made of encapsulated Frankia: assessment of Frankia growth within alginate beads

The growth of Frankia cells within alginate beads was inhibited when the amount encapsulated exceeded 0.5 to 2.5 μg protein/ml of beads. Frankia growth was observed not only in the beads incubated in nutrient media (with of without combined N), but also in those incubated in air provided they retained enough nutrients. The results allow some recommendations to be made for the preparation of Frankia inoculants.

Response of nitrogenase to altered carbon supply in a Frankia‐Alnus incana symbiosis

To study the effect of altered carbon supply on nitrogenase (EC 1.7.99.2), plants of Alnus incana (L.) Moench in symbiosis with the local source of Frankia were exposed to darkness for 2 days, and then returned to normal light/dark conditions. During the dark period nitrogenase activity in vivo (intact plants) and in vitro (Frankia cells supplied with ATP and reductant), measured as acetylene reduction activity, was almost completely lost. Western blots for both the Fe‐protein (dinitrogenase reductase) and the MoFe‐protein (dinitrogenase) showed that, in particular, the amount of MoFe‐protein was strongly reduced during darkness. Protein stained sodium dodecyl sulphate‐polyacrylamide gels of Frankia protein showed that the nitrogenase proteins were the only abundant proteins that clearly decreased during darkness. During recovery, studied for 4 days, nitrogenase activity in vivo recovered to the level before dark treatment but was still only half of control activity, Nitrogenase activity in vitro and the amount of MoFe‐protein, both expressed per Frankia protein, recovered and reached similar values in previously dark treated plants and in control plants. The rate of recovery was similar to the increase in activity of control plants, suggesting growth of Frankia in addition to synthesis of nitrogenase proteins during the recovery after carbon starvation.

Rapid, exponential growth and increased biomass yield of someFrankia strains in buffered and stirred mineral medium (BAP) with phosphatidyl choline

Rapid, exponential growth ofFrankia sp. BR (ORS 020608) isolated fromCasuarina equisetifolia was found to require stirring (magnetic bar) and adequate buffering (MES or MOPS buffers). We used a mineral medium (BAP) at 28°C with propionate and NH4+ as the sole carbon and nitrogen source respectively. Sporulation was enhanced by stirring, but the addition of phosphatidyl choline was found to impair sporulation and slightly improve growth. In these conditions a generation time of 14–16 hours was observed with a maximal biomass yield of 108 μg of protein × mL-1 (Bicinchoninic acid) at day 4 when growth ceased. In stirred conditionFrankia growth as microcolonies (30–200 μm diameter) showing an ‘open mesh’ type of mycelium and also a random distribution of growing hyphae inside and in the periphery of the microcolonies (acridine orange vital staining). In static conditions macrocolonies and only peripheric growth can be observed. Optimal growth in buffered and stirred conditions in mineral medium has been also observed with Cj and CeFFrankia isolates. The reasons for rapid and balanced hyphal growth in stirred conditions are discussed.