Plant and Soil 176: 283-288, 1995. 283 (~) 1995 Kluwer Academic Publishers. Printed in the Netherlands.
Effects of extracts ofAlnus glutinosa seeds on growth of Frankia strain ArI3 under static and fermentor culture conditions
Einar Ring 1,3, Einar Clausen 2, Erik LCvaas 1 , Marijke Van Ghelue I and Bjcrn Solheim 1,4 l lnstitute of Biology and Geology and eThe Norwegian College of Fishery Science, University of Troms, N-9037 TromsO, Norway. 3Present address: Institute of Arctic Veterinary Medicine, Norwegian College of Veterinary Medicine, N-9005 TromsO, Norway. 4Corresponding author*
Received 14 October 1994. Accepted in revised form 22 February 1995
Key words: Alnus glutinosa, cultures, Frankia, seed extract
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.
Members of the genus Frankia are characterized by the ability to form nitrogen-fixing nodules on the roots of a diverse group of woody plants (Baker and Mullin, 1992). However, Frankia grow slowly. As a conse- quence, low biomass production is frequently observed (Blom, 1981, 1982; Blom et al., 1980; Blom and Harkink, 1981; Perradin et al., 1983; Quispel et al. 1983). In static cultures, growth periods of up to 4 weeks were needed to obtain maximum amount of biomass (Blom et al., 1980). Murry et al. (1984) found increased growth rate when cultures were mechani- cally stirred and aerated, but biomass production was low. Enhanced Frankia growth rate and biomass pro- duction were acquired by mechanically stirring of the culture inside the growth vessels in a fermentor (Steele et al., 1989), or with a cross-bar magnet (Schwencke, 1991). Additional improvement of growth was estab-
lished by adding soybean phosphatidyl choline or egg yolk phosphatidyl choline to the medium (Girgis and Schwencke, 1993; Schwencke, 1991).
Plant root exudates and seed extracts have been shown to influence growth and/or development of sym- biotic microorganisms (Hartwig et al., 1991). Such effects have not been demonstrated with Frankia. Some common plant phenolics tested on Frankia inhibited rather than stimulated growth (Perradin et al., 1983). However, lipids extracted from plant roots are found to be necessary in the medium for the isolation and growth of some but not all strains of Frankia (Quis- pel et al., 1983). The active factor in root extract has been identified as the triterpene dipterocarpol (Quispel et al., 1989).
In this paper, the influence of Alnus glutinosa (L.) Gaertn. seed extract on the growth of Frankia strain ArI3 cultured in static and fermentor culture conditions are reported.
* Fax no: + 47 77645600
Materials and methods
Organisms, growth medium and inoculation
Frankia strain ArI3, isolated from nodules of Alnus rubra Bong (Berry and Torrey, 1979), was obtained from D L Akkermans (Agricultural Univer- sity, Wageningen, The Netherlands). Alnus glutinosa (L.) Gaertn. seeds were collected in the Schelde Valley (Wortegern-Petegem, Belgium, December 1990 and 1991). The Frankia growth medium (Meesters et al., 1985) was modified by adding soybean phosphatidyl choline as described by Schwencke (1991).
Precautions were necessary when the medium was prepared for fermentor studies to avoid precipitation, degradation of vitamins and possible loss of propionic acid through evaporation. Mg and Ca salts were auto- claved in 10 L tap water for 1 h at 121 C. After cooling to 27 C, the other components were added separately after sterile filtration through a non-pyrogenic sterile filter (0.22 #m, Sterivex-GS, Millipore). The complete medium was adjusted to pH 5.8 with 6 N NaOH.
A two-week-old culture of actively growing Frankia strain ArI3 was used as inoculum. Frankia mycelium was homogenized by repeated passages through sterile syringe needles (0.8 x 30 mm and 0.6 x 30). Fresh media were inoculated with Frankia to an initial protein concentration of approximately 40 #g protein mL- 1.
Frankia strain ArI3 cultures were not homogenised prior to inoculation of the fermentor. The protein con- centration of fermentor starter cultures was approxi- mately 8 #g mL -1.
Preparation of seed extract
Twenty g seeds ofA. glutinosa were extracted with 100 mL 50% ethanol in water (v/v) with continuous stirring for 18 h. The extract was filtered through a Whatman filter No. 1 and evaporated to dryness at reduced pres- sure. The extracted material was redissolved in 40 mL sterile water.
Growth condition and parameters
Because Frankia has a mycelial growth pattern, cell number cannot be used as measurement of growth. Increase in total amount of protein mL- 1 culture was therefore used as the growth parameter. Frankia was harvested by centrifugation at 8000 g for 10 rain and washed with an excess of sterile water. Prior to protein
analysis (Lowry et al., 1951), the mycelium was boiled in 0.2 M NaOH for 10 rain and sonicated for 45 s at 150 W.
Frankia cultures were examined under a light microscope in order to detect possible contamination in the culture and to determine the size of colonies formed by Frankia hyphae. Frankia colonies are referred to as microcolonies when their size is less then 200 ~tm and as macrocolonies if they are larger.
Na-propionate was the sole carbon source in the medium. The concentration of propionate was deter- mined by isothermal (170 C) analysis in a Hewlett Packard 5880 gas chromatograph, fitted with a NUKOL 30 m capillary column (Supelco). Helium with a flow rate of 25 cm s- 1 was used as the mobile phase. Acetic acid was used as an internal standard.
Frankia biomass was determined by filtering cul- ture samples, each of 100 mL, through 0.45/~m filters (Millipore). Subsequently, these filters were dried at 80 C for 24 h. Dry weight of five parallel samples was used to determine biomass of Frankia. The efficiency of converting carbon source to biomass was calculated as g dry weight cells g- 1 substrate carbon.
Frankia was grown at 27 C either in ! 00 mL Erlen- meyer flasks containing 50 mL of culture medium at static conditions, or in a 10 L Biostat V fermentor (Braun Melsungen AG). Frankia cultures were stirred within the fermentor (200 rpm), and the lower oxygen limit was set at 50% saturation. In experiments includ- ing A. glutinosa seed extract, 5 mL seed extract L- l medium was added.
All growth experiments were repeated three times under the same conditions, and produced similar results.
Growth of Frankia strain Arl3 in static culture
Under static condition, without seed extract, the pro- tein content increased from approximately 40 #g mL- l to approximately 113 ~tg mL -~ over a period of 30 days with a doubling time of 12 days (Fig. 1). When A. glutinosa seed extract was added to the medium the doubling time was reduced to 6 days and at the end of the experiment there was 199/.tg protein mL -1 . Increasing the amount of seed extract in the medium to 10, 20 or 40 mL L -1 medium did not improve growth rates (results not shown). Frankia strain ArI3 cultured in the defined medium with seed extract, but without
c ~ 100 . / o J
ee Q 4) - ~ -4 : [ /
0 5 10 15 20 25 30 35
Fig. 1. Growth of Frankia strain Arl3 measured as protein concen- tration in #g mL- 1 in a defined medium (11) with Na-propionate as carbon source, (A) with 5 mL seed extract L-1 medium added, (O) minus Na-propionate plus 5 mL seed extract L- ] medium. All cul- tures were grown in 100 mL Erlenmeyer flasks with 50 mL medium in static cultures. Each data point represents a mean of two protein determinations.
/ / / /
/ / ~ l l -
LJ E .~ 100
0 .__ L____~_~__ , ' _ __ t _ 0 .00
0 2 4 6 8 16
III ~ \ . / "~ /
10 12 14
0.50 . g
Fig. 2. Growth of Frankia strain Arl3 measured as protein con- centration in #g mL -1 in a fermentor in a defined medium (O) with Na-propionate as sole carbon source, and consumption of Na-propionate (11) during growth. Each data point represents a mean of two protein or three propionate determinations.
Na-propionate did not show any growth (Fig. 1). Inde- pendent of the culture conditions, all the static cultures described above formed only macrocolonies consisting of Frankia hyphae with sporangia and vesicles being absent.
Growth of Frankia strain ArI3 in a fermentor
Frankia strain ArI3 cells were also grown in a Braun Melsungen fermentor, with or without seed extract added to the medium. Under fermentor conditions, Frankia strain ArI3 cultures exhibited doubling times
of 2.5 days based on regression analyses (R=0.99) of log transformed values for protein concentrations from Figure 2. In comparison with static cultures, both growth rates and biomass accumulation were improved in fermentor cultures. In fermentor cultures, the protein content was increased 19-fold whereas in static cul- tures, only a 3-fold increase of starting protein concen- tration occurred. Due to stirring (200 rpm) in fermentor cultures, Frankia mycelium was continuously homog- enized and only microcolonies were observed.
Adding seed extract to Frankia strain ArI3 cultures in a fermentor did not result in enhanced growth rates nor in increased accumulation of biomass. Biomass yield of approximately 150 /~g protein mL -1 and 270-4-20 mg dry weight L -1 were obtained after 14 days at which time close to 90% of the carbon source was used (Fig. 2). The ratio of cell dry weight per gram of substrate carbon source was 0.72.
Duplicate culture samples (150 mL) were taken during the exponential growth phase. One sample was transferred to a 250 mL Erlenmeyer flask and sub- sequently grown under static growth conditions. The other sample was transferred to completely anaerobic conditions. In the subculture transferred to static con- ditions, the growth rate dropped immediately, but fur- ther slow growth was sustained. On the other hand, the anaerobic subculture did not show any further growth (results not shown) which confirms that 02 is necessary for growth. Within two weeks of transfer from fermen- tor cultures to static cultures, microcolonies grew into macrocolonies.
In a separate experiment, the carbon source (Na- propionate) was added to the growth medium accord- ing to Table 1. To keep nitrogen deficiency from limit- ing Frankia growth, an extra 0.2 g NH4C1 was added on day 10. The addition of carbon source was stopped at' day 15 when the Na-propionate concentration was 1.06 g L - ]. During further growth, the concentration of Na-propionate rapidly decreased to 0.06 g L- i at day 20 (Table 1). The growth of Frankia was exponen- tial throughout the experiment when the carbon source was added to the growth medium according to Table 1 (Fig. 3). After 20 days the Frankia culture was harvest- ed and protein content and biomass were determined. A total protein concentration of 241,5 mg L 1 and a biomass of 513+ 15 mg dry weight L - t was measured. The ratio of cell dry weight per gram of substrate car- bon source was 0.75. The doubling time of the biomass of the Frankia culture under these growth conditions was 3.8 days. This was based on regression analyses (R=0.95) of log transformed values for protein con-
Table 1. Amounts of Na-propionate added in g L-l (cumulative values) and actual concentration in the medium during growth of Frankia strain Arl3 in the fermentor
Time in days Cumulative amounts Actual concentration after inoculation Na-propionate added (g L- l) Na-propionate (g L- 1 )
0 0.10 0.18 3 0.30 0.33 6 0.60 0.7 l 9 1.00 0.82
12 1.44 0.96 15 2.00 1.06 18 2.00 0.55 19 2.00 0.22 20 2.00 0.06
3 . . . . . . . . . .
y = 0.779 + 0.096x R = 0.95
C21 ~" .....
0 __ J _~_____L___~L_ ~ . . . . . __ & _ ] 0 2 4 6 8 10 12 14 16
Fig. 3. Growth of Frankia strain ArI3 measured as protein con- centration in/zg mL- 1 in a fermentor in a defined medium. Values have been log transformed. Na-propionate, the sole carbon source, was added to the medium according to Table 1. during growth. The result of the regression analysis of the data is shown. Each data point represents a mean of two protein determinations.
centrations (Fig. 3). The relationship between protein produced and Na-propionate consumed was directly proportional (R=0.97), except for the last day when the propionate was exhausted.
The results presented in this study showed that Frankia strain ArI3, under the applied static growth conditions, exhibits slow growth (doubling time of 12 days). How- ever, the addition of A. glutinosa seed extract to the
medium enhanced the growth yield by approximate- ly 76% and the culture doubling time was reduced to 6 days. No Frankia growth was found in media with seed extract, without Na-propionate. This excludes the possibility that A. glutinosa seed extract could be used as an alternative single carbon source. It is difficult to compare these growth rates with previous results in the literature since media and method of measuring growth varied, but growth periods of 20-30 days are normally needed to reach stationary phase (Burggraaf and Ship- ton, 1983; Perradin et al., 1983; Tisa et al., 1983). Static growth conditions, with or without A. glutinosa seed extract, produced only macrocolonies and shaking the culture, instead of stirring, facilitated even larger aggregates. According to Schwencke (1991), growth is restricted in macrocolonies to peripheral hyphae, which results in the observed slow growth rates.
Quispel et al. (1983) reported that an addition to the media of root extract lipids, later to be identified as the triterpene dipterocarpol (Quispel et al., 1989), was necessary to isolate some strains of Frankia. In a period of adaptation to defined media, root-extracted lipids stimulated the Frankia growth rate. They suggested that the extract may be an excellent carbon source, and in addition might have more specific effects on processes such as membrane synthesis. Dipterocarpol may be incorporated in the membrane of endophytic Frankia, and a gradual adaptation to an environment without dipterocarpol may be needed when transferred to culture media (Quispel et al., 1989). The character of the inducing factors in A. glutinosa seed extract was not investigated in this study, but in contrast to triterpenes it was readily soluble in water and its effect on growth
was found on an isolate well adapted to growth outside its host.
When Frankia strain ArI3 was grown in a fermen- tor, with stirring and adequate aeration, the biomass yield and growth rate improved compared to static cul- tures. In the fermentor a doubling time of about 2.5 days was obtained in contrast to 6 days in a static cul- ture. Previous studies (Murry et al., 1984; Schwencke, 1991; Steele et al., 1989) with stirred cultures also showed enhanced Frankia growth rates. Murry et al. (1984) found a doubling time for Frankia strain Arl3 of 48 h with low biomass production while Schwencke (1991) and Steele et al. (1989) found a doubling time of about 15 h with high biomass production for some fast growing Frankia strains. Differences in observed growth rates might be due to the specific Frankia iso- lates used or to slightly different growth media used in these studies. The enhanced growth of Frankia strain ArI3 cultured in a fermentor could not be improved by adding A. glutinosa seed extract. The major differences between static and fermentor culture conditions are oxygen concentration and size of hyphal aggregates. In fermentor culture conditions with microcolonies more of the mycelium is exposed to the medium and oxygen is abundant. In static culture condition macrocolonies are formed and oxygen might be limited. A. glutinosa seed extract may increase nutrient and oxygen uptake under limiting conditions in static cultures. The mech- anism of growth stimulation is difficult to explain with- out more knowledge of the chemical structure of the active component(s) in seed extract.
It has been shown that increasing the substrate con- centration results in decreased efficiency of converting carbon source to biomass (Steele et al., 1989). By adding the carbon source gradually during growth, the exponential growth period for Frankia cultures could be extended with a high efficiency of converting carbon source to biomass. Direct measurements of propionate concentrations in the medium during growth and indi- rect estimation of cell mass, suggested that propionate is the main growth-yield limiting factor after 14 days when the medium contained 1 g L -~ Na-propionate as an initial carbon source. When Na-propionate was added gradually to a total of 2 g L - l, a growth yield of 5135:15 mg L-1 dry weight and a protein concentra- tion of 241.5 mg L- l was obtained after 20 days.
In conclusion, we found an increased growth of Frankia strain ArI3 in static cultures when A. glut# nosa seed extract was added to the medium. This stim- ulation of growth was not found under stirred, con- trolled growth conditions in a fermentor. Following
the Frankia growth and the carbon source in fermentor grown cultures, we found that under these conditions propionate was the main limiting factor for biomass accumulation. Due to the improved biomass accumu- lation and the production of a homogenous hyphae cul- ture under these conditions, fermentor cultures will be useful in studies concerning physiology and biochem- istry of Frankia and early symbiotic signal exchange in actinorhizal symbioses.
Our results cannot be extrapolated to the soil envi- ronment, but seed and root exudates certainly have a major influence on microbial populations in the rhi- zosphere. As suggested for seed released flavonoids (Hartwig et al., 1991; Phillips et al., 1993), seed exu- date may play a critical role in structuring the microbial community around developing roots in the first hours of germination. A stimulation of growth of Frankia, might be important for the initial infection of the host root.
We are grateful for excellent technical help from Anne Albertsen, Eli Robertsen and Coby Weber. We thank Drs T V Bhuvaneswari, Olavi Junttila and Mike Trinick for critical reading of the manuscript. This work was financially supported by The Norwegian Research Council (Grant 431.403) and the European Commission (Grant SC 1000388)
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