Plant Soil (2014) 384:413–431
DOI 10.1007/s11104-014-2186-6
METHODS PAPER
The art of isolating nitrogen-fixing bacteria
from non-leguminous plants using N-free semi-solid media:
a practical guide for microbiologists
José Ivo Baldani & Veronica Massena Reis &
Sandy Sampaio Videira & Lúcia Helena Boddey &
Vera Lúcia Divan Baldani
Received: 18 December 2013 / Accepted: 24 June 2014 / Published online: 25 July 2014
# Springer International Publishing Switzerland 2014
Abstract
Background and aim Nitrogen-fixing bacteria or
diazotrophs have been isolated for many years using
different formulations of N-free semi-solid media. How-
ever, the strategies used to isolate them, and the recipes
of these media, are scattered through the published
literature and in other sources that are more difficult to
access and which are not always retrievable. Therefore,
the aim of this work was to collate the various methods
and recipes, and to provide a comprehensive methodo-
logical guide and their use by the scientific community
working in the field of biological nitrogen fixation
(BNF), particularly with non-leguminous plants.
Responsible Editor: Euan K. James.
J. I. Baldani (*) : V. M. Reis : S. S. Videira : L. H. Boddey :
V. L. D. Baldani
Embrapa Agrobiologia,
BR 465 km07, Rio de Janeiro, Seropédica 23891-000, Brazil
e-mail: ivo.baldani@embrapa.br
V. M. Reis
e-mail: veronica.massena@embrapa.br
S. S. Videira
e-mail: sandyvideira@yahoo.com.br
L. H. Boddey
e-mail: boddeylh@hotmail.com
V. L. D. Baldani
e-mail: vera.baldani@embrapa.br
S. S. Videira
Centro Universitário de Volta Redonda (UniFOA),
Rio de Janeiro, Volta Redonda CEP 27240-560, Brazil
Methods Procedures used for bacterial counting and
identification either from rhizosphere soil or on the
surface of, or within, plant tissues (to access “endophyt-
ic” bacteria) are presented in detail, including colony
and cell morphologies. More importantly, appropriate
recipes available for each N-free semi-solid culture me-
dium that are used to count and isolate various
diazotrophs are presented.
Results It is recognized by those working in the field of
BNF with non-legumes that the development of the N-
free semi-solid medium has allowed a tremendous ac-
cumulation of knowledge on the ecology and physiolo-
gy of their associated diazotrophs. At least 20 nitrogen-
fixing species have been isolated and identified based on
the enrichment method originally developed by
Döbereiner, Day and collaborators in the 70’s. In spite
of all the advances in molecular techniques used to
detect bacteria, in most cases the initial isolation and
identification of these diazotrophs still requires semi-
solid media.
Conclusions The introduction of the N-free semi-solid
medium opened new opportunities for those working in
the area of BNF with non-legumes not only for eluci-
dating the important role played by their associated
microorganisms, but also because some of these bacteria
that were isolated using semi-solid media are now being
recommended as plant growth-promoting inoculants for
sugarcane (Saccharum sp.), maize (Zea mays) and
wheat (Triticum aestivum) in Brazil and other countries.
Further progress in the field could be made by using a
combination of culture-independent molecular commu-
nity analyses, in situ activity assessments with probe-
414
directed enrichment, and isolation of target strains using
modified or standard semi-solid media.
Keywords Nitrogen fixation . Media recipes . Isolation
procedure . Bacterial counting . Phenotypic
characterization
Introduction
The development of the acetylene reduction (AR) tech-
nique to evaluate nitrogenase activity in the 1960s
(Dilworth 1966; Schöllhorn and Burris 1966) and its
subsequent application to investigate BNF associated
with non-legume crops (Yoshida and Ancajas 1970;
Balandreau and Dommergues 1971; Döbereiner et al.
1972) generated great interest in the concept that non-
legumes may be able to benefit from N inputs from this
process. Without doubt, one of the most important pio-
neers in the area of BNF associated with non-legume
plants was Dr Johanna Döbereiner, the founder of our
institute, now known as Embrapa Agrobiologia. Since
she started work at the research institute at Km 47 in
Seropédica, Rio de Janeiro, in 1951, she showed a great
interest in N2-fixing (or diazotrophic) bacteria isolated
from rhizosphere soil (Döbereiner 1953) or associated
with sugarcane (Döbereiner and Ruschel 1958) and
other grasses, such as Paspalum notatum (Döbereiner
1966). Up until her death in 2000, the team she headed
discovered and named nine new species of diazotrophs
and had developed new techniques to isolate and iden-
tify these bacteria (Baldani and Baldani 2005). Even
now, more than 10 years after her passing, all the differ-
ent techniques that she and her team developed are
scattered across the literature and many of these publi-
cations are from the pre-electronic (PDF) period, some
are only in Portuguese, and many of them are difficult to
access. The interest in the bacteria which associate,
sometimes endophytically, with non-nodulating plants
continues unabated. For just the keyword
“Azospirillum”, the best known nitrogen-fixing and
plant growth-promoting genus, the Web of ScienceTM
lists over 3199 publications in all databases since its
discovery in 1978 until 2014 (updated March 17,
2014). The accumulated literature, especially on
Azospirillum, indicates that in addition to BNF, princi-
pally in association with sugarcane (Oliveira et al.
2006), these bacteria also benefit host plants through
other mechanisms, such as phytohormone and
Plant Soil (2014) 384:413–431
siderophore production, P-solubilization and biological
control of phytopathogens (Santi et al. 2013; Compant
et al. 2010).
Many researchers require in their work to isolate and
identify these N2-fixing bacteria and, therefore, the ob-
jective of this guide is to put together most of the
available information related to the strategy for isolation
and identification of diazotrophs, not only those more
frequently studied by the team of Johanna Döbereiner,
but also by other authors both contemporaneous and
more recent who have applied the semi-solid medium
to survey the occurrence of N2-fixing bacteria in their
agricultural systems and climatic conditions.
Development of the semi-solid medium
Until the beginning of the 1970’s, reports on N2-fixing
bacteria referred only to those bacteria able to grow
under atmospheric oxygen concentration (pO2 =0.21
kPa). However, the key enzyme responsible for the
reduction of the N2 gas in the atmosphere, the nitroge-
nase complex, is highly sensitive to oxygen and may
induce irreversible damage to its activity (Dalton and
Postgate 1968). Media used to isolate nitrogen-fixing
bacteria from the environment were normally prepared
in a solid form and only a few diazotrophic bacteria
were described at that time using this traditional ap-
proach, such as Beijerinckia fluminensis, Derxia
gummosa, D. indica and Azotobacter paspali (Baldani
and Baldani 2005). Also, most of the rhizobia were
isolated using only a solid medium, usually Culture
Medium No. 79 of Fred and Waksman (1928) otherwise
known as yeast mannitol agar (YMA) (Vincent 1970).
The so-called “semi-solid” medium was initially tested
for different purposes (for details - see Hitchens 1921)
and later applied, for example, to measure the reduction
of nitrate to nitrite (ZoBell 1932), to detect the oxidation
of carbohydrates and distinguish it from fermentation by
various Gram negative bacteria (Hugh and Leifson
1953), and to study bacterial chemotaxis (Adler 1966).
The concentration of agar in all these semi-solid media
varied from 2 to 3 g L−1, which was considerably higher
than that defined for the N-free semi-solid medium used
for isolating N2-fixing bacteria in the 70’s by Döbereiner
and Day (1976). This agar percentage difference be-
tween these semi-solid media may be supported by the
work of Whittenbury (1963) who developed what he
called “soft agar medium”. This author applied the
Plant Soil (2014) 384:413–431
medium containing 1.5 g L−1 agar to study lactic acid
bacteria and concluded that this soft agar provided a
range of environments from aerobic to anaerobic and
thus permitted the inoculum to develop in that region of
the medium most suitable for it. This is exactly the
behavior observed for the vibrio-like organisms with
very characteristic corkscrew movements, such as the
Spirillum lipoferum described by Beijerinck 1925, and
later reclassified in the newly-created genus
Azospirillum (Tarrand et al., 1978). According to
Döbereiner and Pedrosa (1987), the introduction of the
N-free semi-solid medium, such as NFb medium in the
case of Azospirillum (see later for details), was the key
which allowed for the isolation and identification of
many nitrogen-fixing bacteria associated with poaceous
plants.
The N-free semi-solid medium was originally con-
sidered to be a minimal enrichment medium used to
improve the growth of N2-fixing bacteria at the expense
of non- N2-fixing bacteria. No nitrogen source was
added to the recipe and the pH was adjusted to the
optimal level for each species described based upon
high acetylene reduction activity (ARA) (Day and
Döbereiner, 1976). Several media were described with
different carbon sources, buffers, mineral concentrations
and the addition, or not, of vitamins. Usually, the semi-
solid medium occupies half of the vial volume (10 mL)
and allows the inoculation of a single drop of sample
(cell, plant tissue or soil suspension) into the center of
the medium. The consistency of the semi-solid agar
medium is crucial for bacterial growth that is dependent
on BNF. The medium should be neither too solid nor too
liquid, and so the amount of agar added (1.4 to
1.8 g L−1) needs to be adjusted every time the chemical
company providing the agar is changed. In addition, the
pH also interferes with the consistency of the medium,
and so care should be taken when preparing more acidic
media, such as JMV and LGIP (see later). To prepare
semi-solid media, in each case the agar must be mixed
with the media compounds, melted and distributed into
vials before autoclaving. In general terms, the principle
of this semi-solid medium is that it allows for growth of
the bacteria under conditions where their nitrogenase
requires protection from O2-mediated denaturation
(Dalton and Postgate 1968) i.e. in early stages of growth
when only a small number of cells are starting to mul-
tiply and they thus use little oxygen. In later stages, the
bacterial cells within the culture medium migrate closer
to the surface where there is sufficient oxygen pressure
415
to support aerobic respiration, but not so much that it
damages nitrogenase. With this procedure plant-
associated diazotrophic bacterial counts can be made
using various plant samples, although it was originally
devised only for root samples. From the use of these
media the Azospirillum genus rapidly acquired descrip-
tions of new species, such as A. amazonense (Magalhães
et al. 1983), A. halopraeferens (Reinhold et al. 1987)
and A. irakense (Khammas et al. 1989).
The original NFb semi-solid medium developed for
Azospirillum by Döbereiner and Day (1976) allowed the
development of other media just by replacing the carbon
source, and changing the pH and osmotic concentration,
omitting and adding vitamins, salts, amino acids,
root/shoot extracts, etc. in order to mimic the environ-
ment or plant of interest. Based on these modifications
new nitrogen-fixing species and even genera were iso-
lated and identified in N-free enrichment cultures as
shown in Table 1.
Counting and isolating diazotrophic bacteria
associated with non-legumes
The serial dilution technique is used to count the number
of cells per unit of sample, but also facilitates the pro-
cedure to isolate N2-fixing bacteria. Considering that the
number of bacteria present in the sample is reduced by
applying serial dilution it is recommended that the vial
with the highest dilution showing positive growth (i.e.
with pellicle formation) is processed for purification and
further identification procedures if the interest is to
isolate the bacterium that is present in high numbers.
This method is also very useful to isolate diazotrophic
bacteria that grow under the same conditions but which
are found in lower numbers. The use of semi-solid
media is considered to be an enrichment method to
assess the population present in the sample in a given
harvested time. However, they also allow a survey of the
diversity of part of the culturable diazotrophic bacterial
population present in the soil or in plant tissues. Figure 1
explains in more detail about the steps used for counting
and isolating diazotrophic bacteria associated with non-
legumes.
Steps for sampling and processing
In the utilization of the Most Probable Number (MPN)
method to count and isolate a fraction of culturable
416 Plant Soil (2014) 384:413–431

Table 1 N-free semi-solid media used to isolate non-symbiotic nitrogen-fixing bacteria

Media Bacterial species Carbon source pH References

NFb Azospirillum lipoferum Malic acid 6.8 Döbereiner and Day (1976)
Azospirillum brasilense 6.8 Döbereiner and Day (1976)
Azospirilum irakense 7.0 – 8.5 Khammas et al. (1989)
Azospirillum doebereinerae Eckert et al. (2001)
Azospirillum melinis 6.5 – 6.8 Peng et al. (2006)
Azoarcus olearus 6.5 – 7.3 Chen et al. (2013)
LGI Azospirillum amazonense Sucrose 6.0 – 6.2 Baldani (1984)
FAM Azospirillum amazonense Sucrose 6.0 Magalhães and Dobereiner (1984)
JNFb Herbaspirillum seropedicae Malic acid 5.8 Baldani et al. (1992)
Herbaspirillum rubrisubalbicans Baldani et al. (1996)
Herbaspirillum frisingense Kirchhof et al. (2001)
JNFb Sphingomonas spp. Malic acid 5.8 Videira et al. (2009)
LGI-P Gluconacetobacter Crystalized cane sugar 5.5 Cavalcante and Döbereiner (1988)
diazotrophicus Reis et al. (1994)
G. johannae Fuentes-Ramírez et al. (2001)
G. azotocaptans
JMV Burkholderia kururiensis Mannitol 5.0 – 5.4 Baldani et al. (2000)
Burkholderia tropica Reis et al. (2004)
Burkhoderia silvatlantica Perin et al. (2006)
Baz Diazotrophic Burkholderia Azelaic acid 5.7 (Estrada-de-los-Santos et al. 2001)
Bac Diazotrophic Burkholderia Azelaic acid+L-citrulline 5.7 (Estrada-de-los-Santos et al. 2001)

diazotrophic bacteria, it is assumed that the bacteria are
released from the soil aggregates and/or plant tissues by
the vortexing/maceration procedure, and that the subse-
quent serial dilutions are based on a homogenous sus-
pension of individual bacterial cells. It is further as-
sumed that single cells of the target organisms can grow
under these conditions, and that other bacteria present in
the suspension will not inhibit their growth in the semi-
solid medium. Hence, it is apparent that the MPN tech-
nique underestimates the population of diazotrophs
present in plant tissues, as was demonstrated by Li and
MacRae (1992) and Silva-Froufe et al. (2009), both of
whom a pplie d the ELI SA m etho d to co unt
Gluconacetobacter diazotrophicus and Herbaspirillum
seropedicae in sugarcane plants growing in the field.
Methods applying oligonucleotide probes coupled to
epifluorescence microscopy (Stoffels et al., 2001; Watt
et al. 2006) or qPCR using strain/species-specific
primers (Pereira 2014) may provide an approximation
of the total diazotrophic population associated with the
non-legumes, although more recent tools like
microbiome studies using 16S rRNA gene sequence
profiling by next generation sequencing techniques
(Turner et al. 2013) or metagenomic studies (Sessitsch
et al., 2012) probably reveal a more complete picture of
the N2-fixing community colonizing the rhizosphere
and internal tissues of poaceae. A drawback of these
latter methods, of course, is that although they are
excellent for examining bacterial diversity, they do not
result in the isolation of bacterial strains that can then be
used for further analyses and/or tested as inoculants.
Nevertheless, the MPN technique has been routinely
used to count and isolate culturable diazotrophic bacte-
ria from different parts and tissues of grasses, such as
rice (Ferreira et al., 2010), maize (Conceição et al.
2009), sugarcane (Reis Junior et al. 2000), forage
grasses (Kirchhof et al. 2001; Brasil et al. 2005;
Videira et al. 2012), and has also been applied to fruits,
such as banana (Musa sp.) and pineapple (Ananas
comosus - Weber et al. 1999), as well as oil palm trees
Plant Soil (2014) 384:413–431
Fig. 1 Diagram showing all the
steps applied to count and isolate
free-living, associative and
endophytic diazotrophic bacteria
(Elaeis guineensis - Carvalho et al. 2006), and many
other plants in many countries. It can also differentiate
the external from the internal bacterial colonization
by applying a surface disinfection procedure in ad-
vance, such as that described by (Baldani et al.
1986a), which is detailed below. In addition, the
MPN method is easy to apply in any laboratory with
a large number of samples, and because it is relatively
inexpensive, the numbers of nitrogen-fixing bacteria
such as those applied as inoculants can be monitored
quite easily and cheaply. One limitation in monitoring
inoculated strains is the difficulty to separate them
from the endogenous diazotrophic population
417
associated with the target plants, and in this case the
application of strain-specific immunological in situ
detection methods (James and Olivares 1998;
Schloter and Hartmann, 1998; James et al. 2001;
Rothballer et al., 2008; Schloter et al. 1995) or quan-
titative PCR techniques (Ruppel et al. 2006) are
desirable.
Rhizosphere soil
Rhizosphere soil (i.e. soil around the roots) can either be
directly inoculated into the N-free semi-solid medium
using a loopful of soil or a sample (1–10 g). These
418
samples are normally used in a mixture with 9–90 mL of
saline solution containing (mg L−1) K2HPO4, 100;
MgSO4, 50; NaCl, 20; CaCl2.2H2O, 50; FeEDTA,
16.4. The addition of Tween 80 (1 g L−1) can be applied
to aid bacterial dispersion in soil samples, as well as in
samples of bacteria mixed with peat, which is often used
as a carrier for bacterial inoculants. Other methods uti-
lize sucrose solution (40 g L−1 in water), especially in
soil samples collected from the rhizosphere of sugar-rich
plants like sugarcane and Miscanthus (Eckert et al.
2001). The tube containing the 10−1 diluted solution
should be capped and shaken vigorously (for ap-
proximately 1 h) until the soil is evenly dispersed
in the saline solution. The bacterial population in
the rhizosphere, a stable environment that is rich in
nutrients, is enormous, usually in the range of 106
to 109 viable bacteria per gram of rhizosphere soil
(Bulgarelli et al. 2013). Therefore, it is generally
suggested to use serial dilutions down to 10−6 or
10−9 to count and isolate free-living N2-fixing bac-
teria, but these numbers vary with soil moisture, so
the environment conditions at the site where the
samples were collected should always be taken into
consideration (Oliveira et al. 2004).
Fig. 2 First and second steps applied to count and isolate
diazotrophic bacteria. a. Sampling and processing plant samples,
b. Serial dilution and inoculation into semi-solid media. I. Sam-
pling of plants; II. Root-free from soil or substrate; III. Root and
Plant Soil (2014) 384:413–431
Plant samples - roots, stems and leaves
Roots removed from field-grown plants should be
washed in tap water to remove rhizosphere soil. They
should then be cut into 10-cm pieces, dried on a paper
towel, weighed and then 10 g samples are blended (at
approximately 3,200 rpm) in 90 mL of the above saline
or sugar solutions for one to 2 minutes (Fig. 2a). The
suspensions should then be left to stand for 30–60 min
after blending which allows the bacteria to migrate from
the plant tissue into the suspension. The suspension is
then agitated to homogenize it for 5 to 10 min on a rotary
shaker (~150 rpm) and then, 10-fold serial dilutions are
made. Several factors, such as host species, genotype
and plant age, as well as the location of the tissues within
the plant can all have an impact on the structure of the
bacterial communities associated with the plants (van
Overbeek and van Elsas 2008; Bodenhausen et al.
2013). Generally, bacterial populations are highest
on root surfaces, followed by root internal tissues,
and then in the aerial tissues. It is commonly rec-
ommended to use serial dilutions down to 10−7 or
10−9 to count and isolate N2-fixing bacteria from
roots and 10−5 or 10−6 from aerial parts.
shoot tissues separated; IV. First dilution of samples (10−1) on
saline solution; V and VI. Serial dilution; VII and VIII. Vortexing
samples before inoculation; IX and X. Inoculation of 0.1 mL into
semi solid media
Plant Soil (2014) 384:413–431
Table 2 Efficiency of maize root surface disinfection with Chlo-
ramine–T
Time of exposure to Treatments ARA nmoles C2H4/h/
Chloramine-T (minutes) of roots culture roots with±8 cm
0 intact 12947
0 crushed 13326
5 intact 846
5 crushed 8631
30 intact 758
30 crushed 1895
60 intact 0 further isolation of the endophytic diazotrophic bacteria
60 crushed 583
- ARA evaluated 42 h after incubation in semi-solid NFb medium
- root tips were immersed in paraffin oil before and after
disinfection
- Adapted from Baldani (1984)
Patriquin and Döbereiner (1978) surface disinfected
roots of maize with a reagent active in chlorine known
as Chloramine-T (CH3. C6H4.SO2.N Na Cl.3 H2O) with
the objective of isolating diazotrophic bacteria from
within the roots. The isolation of N2-fixing bacteria from
within plant tissues using this simple method led to the
concept of “endophytic diazotrophs” (Döbereiner 1992)
based on the definition of Kloepper et al. (1992) who
suggested to replace the term endorhizosphere with the
new term endophytes to refer to microorganisms found
colonizing inside roots or interior tissues. The term
endophyte, which had long been used to refer to (non-
mycorrhizal) beneficial fungi living within plants, was
later also used to refer to all bacteria that are able to
colonize the inner tissues of plants without causing any
apparent damage to the host (Hallmann et al. 1997).
A concentration of 10 g L−1 of Chloramine-T is
recommended for surface disinfection, and the time of
immersion in this solution is determined by the age and
type of plant: Usually roots from maize and sorghum at
the flowering stage are immersed for 30 to 60 min,
whilst those from rice and wheat can only resist 5 to
15 min in the same solution. After surface disinfection,
roots should be placed in sterile distilled water for a 1/3
of the time that they were in the disinfecting solution.
This is repeated for the same time in phosphate buffer
(50 mM, pH 7.0) and again in distilled water, with the
total time amounting to that of the sterilization in Chlo-
ramine T. Roots that are not to be disinfected should be
soaked in distilled water for the same time as that used in
419
the sterilization procedure (Baldani et al. 1986a). The
confirmation of the effectiveness of root surface disin-
fection process can be performed using the methodolo-
gy described by (Baldani et al. 1986b), where surface
disinfected roots are capped with paraffin wax on both
ends and then submerged in 80 mL of the specific semi-
solid medium in large test-tubes. Three days after incu-
bation, roots from tubes showing no bacterial growth
should be crushed within the tubes or transferred to new
semi-solid medium, and then the hopefully newly-
released microorganisms are allowed to grow, so that
present in the internal tissues can be undertaken.
Chloramine-T is effective for roots, but not for aerial
tissues. An example of surface disinfection efficiency is
shown in Table 2 where zero nitrogenase activity was
observed when intact roots that were exposed for 1 hour
in Chloramine-T and capped with paraffin wax were
compared with sterilized crushed roots. A reduction in
the sterilization time led to positive nitrogenase activity
even for capped roots, thus indicating that the duration
of effective surface disinfection is dependent on the
plant type, age and tissue, as has been reviewed by
Hallmann et al. (1997).
In the case of stems e.g. those on sugarcane and
elephant grass (Pennisetum purpureum), it is first nec-
essary to remove dust and waxes from the surfaces with
tap water before proceeding with the disinfection pro-
cedure. The stems are sprayed with 70 % ethanol,
flamed, and then peeled with a knife to remove the
external tissue. To ensure that no bacteria from the plant
surface are present in the stems they are dipped into
70 % ethanol and flamed again. Leaf samples should
first be washed in tap water and then surface disinfested
with 70 % ethanol. For counting and isolation of the
bacteria in leaf samples they should be treated in the
same manner as described above for soil, roots and
stems i.e. 10 g of leaf material are homogenized in
90 mL of saline or sucrose solution followed by 10-
fold serial dilutions (Videira et al. 2012).
Counting procedures
In most cases, the numbers of non-symbiotic nitrogen-
fixing bacteria (free-living, associative and endophytic)
are estimated by the MPN method (Cochran 1950) using
McCrady’s probability tables (Okon et al. 1977). This
classic method is based on extinction of the population
using a serial dilution procedure. Normally, a 10-fold
420
serial dilution in saline or sucrose solution is used, and
samples are assumed to differ in predicted numbers
based upon these dilutions. In order to count the cells,
0.1 mL of each dilution (generally the highest ones -
10−4 up to 10−9) are inoculated into vials containing
5 mL of each N-free semi-solid medium (Fig. 2b). This
step can be performed with 3 or 5 replicates.
Inoculation into semi-solid medium
For most microaerobic diazotrophs the MPN method
using N-free semi-solid media relies upon the appear-
ance of a typical diazotrophic bacterial pellicle in the
subsurface of the medium after incubation for 7–10 days
at 30ºC (Fig. 3). Observation of the initial pellicle for-
mation is usually possible 2–3 days after inoculation and
growth must be then observed every subsequent day as
some bacteria can grow very quickly. Counting can
generally be performed after 5–7 days growth (Fig. 3).
The characteristic bacterial pellicles in vials with the
highest dilution are transferred to a fresh N-free semi-
solid medium, and after confirmation of bacterial
Fig. 3 Inoculation of nitrogen-free semi-solid media and moni-
toring the pellicle formed in the media. a. Veil like pellicle formed
2 days after inoculation. b. Surface/subsurface pellicle formed
7 days after inoculation. The black arrows in figures indicate the
characteristic pellicle of the diazotrophic bacteria during growth in
Plant Soil (2014) 384:413–431
Fig. 4 Characteristics of colony morphology of several„
diazotrophic bacteria grown on different purification media a.
Azospirillum brasilense strain Sp245 on NFb, BMS (Basal Medi-
um with Sucrose) and BDA (Potato Dextrose Agar) b. Azoarcus
olearius on NFb and BDA c. Azospirillum amazonense strain
CBAmC on LGI and BMS d. Herbaspirillum seropedicae strain
HRC54 on JNFb, NFb, BMS and BDA; e. Sphingomonas spp.
strain BR12195 on JNFb, NFb, LGI and BMS f.
Gluconacetobacter diazotrophicus strain PAL5 on LGI-P, Potato-
P and BDA; g. Burkholderia kururiensis strain KP23 and
B.tropica strain Ppe8 on JMV and on BDA
growth, a loopfull of the new pellicle is streaked onto
the corresponding solid semi-specific medium contain-
ing a trace amount of yeast extract (around 40 mg L−1)
to isolate the target bacterium based on the phenotypic
characteristics of the colonies (Döbereiner 1995)
(Fig. 4). A single purified colony is again checked
in the same N-free semi-solid medium and the
flasks that originally contained the characteristic
pellicle are used for isolation of the bacteria and
subsequent purification on potato agar medium
(Baldani et al. 2000) (Fig. 4).
different semi-solid media. I. Azospirillum brasilense in NFb 3x,
II. Herbaspirillum seropedicae in JNFb, III. Azoarcus olearius in
N F b 3 x , I V A z o s p i r i l l u m a m a z o n e n s e i n L G I , V.
Gluconacetobacter diazotrophicus in LGI-P, VI Burkholderia
kururiensis in JMV
Plant Soil (2014) 384:413–431 421
422
Recipes for the N-free semi-solid and solid media
and description of the bacterial colony types forming
on the latter
NFb medium
This medium was initially denominated Fb, after the
researcher Dr. Fabio Pedrosa. After modifications, it
was then denoted NFb (Dobereiner et al. 1976), with
the N meaning “new”. The basic medium contains
(g L−1): malic acid, 5.0; K2HPO4, 0.5; MgSO4.7H2O,
0.2; NaCl, 0.1; CaCl2. 2H2O, 0.02; micronutrient solu-
tion (CuSO4.5H2O, 0.04; ZnSO4.7H2O, 0.12; H3BO3,
1.40; Na2MoO4.2H2O, 1.0; MnSO4. H2O, 1.175. Com-
plete volume to 1,000 mL with distilled water), 2 mL;
bromothymol blue (5 g L−1 in 0.2 N KOH), 2 mL;
FeEDTA (solution 16.4 g L−1), 4 mL; vitamin solution
(biotin, 10 mg; pyridoxal-HCl, 20 mg. Dissolve in hot-
water bath. Complete to 100 mL by adding distilled
water), 1 mL; KOH, 4.5 g; Distilled water to bring the
final volume to 1,000 mL and adjust pH to 6.5. A
quantity of 1.6 to 1.80 g agar L−1 should be added to
prepare the semi-solid medium and 15 g agar L−1 for the
solid medium. It is important that the various ingredients
are added in the given sequence to avoid precipitation of
iron or of other salts due to the high pH.
Overall, A. brasilense and A. lipoferum require inoc-
ulation into semi-solid NFb medium (Fig. 2, Step 5a).
The pellicle should be streaked onto solid NFb, and then
onto BMS (Batata- Malato- Sacarose) agar medium,
also known as Potato medium, or on Bacto Dextrose
agar (BDA) (Fig. 4a). In BMS the colonies formed are
initially yellowish-white becoming pinkish as they be-
come larger and are of wet appearance on BDA (Step
5a). Azospirillum doebereinerae (Eckert et al. 2001) is
also commonly isolated with NFb semi-solid medium
after incubation for 3 to 5 days at 30 °C. Further purifi-
cation is done on NFb (with yeast extract at 50 mg L−1)
and half-strength DYGS medium (modified from that
described by Rodrigues Neto et al. 1986) agar plates. On
NFb agar plates with 50 mg yeast extract and triple-
strength bromothymol blue, the colonies are 0.5 mm in
diameter and appear grey and dull. Colonies grown on
medium with bromothymol blue replaced with congo
red indicator are scarlet (Rodriguez-Cáceres 1982).
The genus Azoarcus (type species, Azoarcus indi-
gens) was proposed by Reinhold-Hurek et al. (1993).
Some species fix nitrogen and then require microaerobic
conditions for growth dependent on N2 (Reinhold-
Plant Soil (2014) 384:413–431
Hurek and Hurek 2006). The newly-described
diazotrophic species, Azoarcus olearius (Chen et al.
2013) is able to grow in semi-solid NFb medium
(Fig. 4b). On NFb agar plates the colonies formed are
white and wet, while on BDA plates the colonies are
large, wet and white (Fig. 4b).
LGI medium (Baldani et al. 1984) and FAM Medium
(Magalhães and Döbereiner 1984)
The LGI medium, which was developed from LG
medium (Lipman, 1904) with the I derived from
“Ivo”, contains (g L−1): sucrose, 5.0; K2HPO4, 0.2;
KH2PO4, 0.6; MgSO4.7H2O, 0.2; CaCl2.2H2O, 0.02;
Na2MoO4.2H2O, 0.002; bromothymol blue (5 g L−1
in 0.2 N KOH), 5 mL; FeEDTA (solution
16.4 g L−1), 4 mL; vitamin solution, 1 mL. Complete
volume to 1,000 mL with distilled water. Adjust pH
to 6.0 to 6.2 with H2SO4 (5 % solution). For semi-
solid medium add 1.6 to 1.80 agar L−1 and 15 g agar
L−1 for solid medium.
The FAM medium contains (g L−1) sucrose, 5.0;
KH2PO4, 0.12; K2HPO4, 0.03; MgSO4.7H2O, 0.2;
C a C l 2 , 0 . 0 2 ; F e E D TA , 0 . 0 6 6 ; N a C l , 0 . 1 ;
NaMoO4.2H2O, 0.002; MnSO4, 0.00235; H3BO3,
0.0028; CuSO 4 .5H 2 O, 0.00008; ZnSO 4 .7H 2 O,
0.00024; biotin, 0.0001; pyridoxine-HCl, 0.0002 g. Dis-
tilled water is added to bring final volume to 1,000 mL.
For semi-solid medium add 1.6 g agar L−1 and adjust pH
to 6.0.
Azospirillum amazonense (Magalhães et al.
1983) is best isolated in a semi-solid sucrose–based
medium, such as LGI (Fig. 4c) or FAM, in the
same way as described above. Sub-surface pellicles
formed after 3 to 5 days incubation at 35 °C are
transferred to fresh semi-solid medium and are
again incubated (Fig. 4c). Then the pellicles are
streaked onto solid LGI with 50 mg L1 yeast ex-
tract where colonies appear small, whitish, curled
with a firm dense, but not tenacious, consistency
and partially embedded into the agar (Fig. 3b).
Individual colonies are transferred into semi-solid
LGI medium and then can be streaked onto plates
of potato agar (with sucrose as the sole carbon
source) where they will grow as large, flat white
colonies (5 mm) with raised margins (Fig. 4c).
Colony morphology may be altered when different
C-sources are used in the media (for details see
Baldani et al. 2005a).
Plant Soil (2014) 384:413–431
JNFb medium (Baldani et al. 1992)
Developed by J. Döbereiner from NFb medium
(Baldani et al. 1992), the JNFb medium contains
(g L−1) malic acid, 5.0; K2HPO4, 0.6; KH2PO4, 1.8;
MgSO4.7H2O, 0.2; NaCl, 0.1; CaCl2.2H2O, 0.02; Mi-
cronutrient solution (above), 2 mL; bromothymol blue
(5 g L−1 in 0.2 N KOH), 2 mL; FeEDTA (16.4 g L−1),
4 mL; vitamin solution, 1 mL; KOH, 4.5. Add distilled
water to bring total solution to 1,000 mL. Adjust the pH
to 5.8 with KOH. To semi-solid and solid medium add
1.8 and 17 g agar L−1, respectively.
Herbaspirillum seropedicae, H. rubrisubalbicans and
H. frisingense (Baldani et al. 1987; 1996; Kirchhof et al.
2001) form a veil-like pellicle in JNFb semi-solid medium
similar to that of Azospirillum spp. (Fig. 4d). These bacte-
rial pellicles are generally streaked onto JNFb or NFb agar
plates containing yeast extract (50 mg L−1) where colonies
become small and white with a central blue point after
1 week incubation (Fig. 4d). This color is more evident in
NFb medium with three times the normal concentration of
bromothymol blue (denoted NFb 3x) mainly for strains of
H. seropedicae and H. rubrisubalbicans (for details see
Baldani et al. 2005b). Purification on potato medium with
sucrose and malate yields small wet raised colonies which
become brownish in the center while they remain white
and wet on BDA (Fig. 4d).
Recently, nitrogen-fixing strains in the genus
Sphingomonas were isolated using JNFb medium
(Videira et al. 2009). The procedure for isolation and
purification is the same as for Herbaspirillum species,
but the incubation period is longer than 7 days. Howev-
er, in contrast to Herbaspirillum species, white pellicles
are initially formed which later become yellow with loss
of the blue color of the JNFb medium (Fig. 4e). This
characteristic is considered presumptively to be positive
growth of Sphingomonas rather than Herbaspirillum. In
addition, light green Sphingomonas colonies are formed
on JNFB agar medium, but they appear yellow on potato
agar medium (Fig. 4e).
LGI-P medium (Reis et al. 1994)
Based on the LGI medium (Baldani, 1984), LGI-P was
developed to isolate and count G. diazotrophicus from
sugarcane plants. The letter P stands for the state of
Pernambuco in Brazil, the place where
G. diazotrophicus was first isolated. The composition is
(g L−1): crystallized cane sugar, 100; K2HPO4, 0.2;
423
KH2PO4, 0.6; MgSO4.7H2O, 0.2; CaCl2.2H2O, 0.02;
Na2MoO4.2H2O, 0.002; bromothymol blue (5 g L−1 in
0.2 M KOH), 5 mL; FeCl3.6H2O, 0.01. Add distilled
water to bring total solution to 1,000 mL. Adjust the pH
to 5.5 using acetic acid. Add 1.8 g and 17 g agar L−1 for
semi-solid and solid medium, respectively.
Gluconacetobacter diazotrophicus (originally named
Saccarobacter nitrocaptans - Cavalcante and Döbereiner,
1988), G. azotocaptans and G. johannae (Fuentes-
Ramírez et al. 2001) are able to grow in semi-solid LGI-
P medium (Fig. 2, Step 5) containing 100 g L−1 of crystal
sugar (10 %). LGI-P medium with 5 mL L−1 of sugarcane
juice is called LGI-Pc and is used to recover
G. diazotrophicus from plant samples, especially from
sugarcane (Reis et al. 1994). Seven to ten days after
inoculation of diluted samples into semi-solid LGI-Pc,
the vials show orange pellicles and the medium becomes
colorless (Fig. 4f). The pellicle is then streaked onto plates
containing solid LGI-P medium amended with 50 mg L−1
yeast extract, and incubated for 7 days at 30 °C. Colonies
of G. diazotrophicus are small and orange (Fig. 4f). The
colonies of G. johannae are yellow-orange, very irregular,
smooth and flat after 5 d growth. The G. azotocaptans
colonies are orange but form round, mucoid, smooth and
convex colonies with translucent margins. The purifica-
tion step is made by streaking colonies onto Potato-P
medium, where colonies of G. diazotrophicus are initially
moist and clear, changing to chocolate brown 7 to 10 days
after incubation at 30 °C (Fig. 4f).
JMV (Baldani, 1996), BAz and BAc media
(Estrada-de-los-Santos et al. 2001)
The JMV medium contains (g L−1) mannitol, 5.0;
K2HPO4, 0.6; KH2PO4, 1.8; MgSO4.7H2O, 0.2; NaCl,
0.1; CaCl2.2H2O, 0.2; bromothymol blue (5 g L−1 in
0.2 N KOH), 2 mL; FeEDTA (16.4 g L−1), 4 mL; Mi-
cronutrient solution (see above), 2 mL; vitamin solution,
1 mL. Add distilled water to bring total solution to 1,000
mL. Adjust the pH to 5.0 - 5.4 with KOH. To semi-solid
and solid medium add 1.8 and 25 g agar L− 1 respectively.
Yeast extract (0.1 g) can be added in semi-solid medium
to stimulate growth of pure culture when evaluated in
laboratory conditions.
BAz medium has the following composition (g L−1):
azelaic acid, 2.0; K2HPO4, 0.4; KH2PO4, 0.4; MgSO4 ·
7H2O, 0.2; CaCl2, 0.02; Na2MoO4 ·H2O, 0.002; FeCl3,
0.01; bromothymol blue, 0.075; and agar, 2.3. Adjust the
pH to 5.7 with KOH. Add distilled water to bring total
424
solution to 1,000 mL. Vials containing 5 ml of BAz
medium are autoclaved at 121 °C for 20 min, and filter-
sterilized cycloheximide (200 μg/tube) is then added.
The BAc medium contains (g L−1): azelaic acid, 2.0;
L-citrulline, 0.2; K2HPO4, 0.4; KH2PO4, 0.4 and
MgSO4 ·7H2O, 0.2. Adjust the pH to 5.7 with KOH
and the medium is sterilized at 121 °C for 20 min prior
to the addition of filter-sterilized (pore size, 0.22 μm)
citrulline as the sole nitrogen source. Add distilled water
to bring total volume to 1,000 mL.
Nitrogen fixing species, such as the Brazilian
Burkholderia kururiensis strains (Baldani and Baldani
2005) as well as the species B. tropica (Reis et al. 2004),
B. silvatlantica (Perin et al. 2006) and B. unamae (Cabal-
lero-Mellado et al. 2004) are easily isolated applying the
JMV medium containing mannitol as a carbon source.
However, the BAz and BAc media were also used by Reis
et al. (1994) and Caballero-Mellado et al. (2004) to isolate
and cultivate these Burkholderia species. Other recipes are
also described by (Estrada-de-los-Santos et al. 2001) to
isolate new species of Burkholderia from plant samples.
Bacteria of this genus are very versatile and can also grow
in LGI and LGI-P medium. The serial dilutions are inoc-
ulated into the semi-solid medium and incubated 4 to
7 days at 32 °C. On the fourth day thick pellicles will
begin to form (Fig. 4g). Once these pellicles have migrat-
ed to the surface of the medium, they are streaked onto
plates of JMV solid medium amended with 60 mg L−1
yeast extract and again incubated for 4 to 7 days at 32 °C.
Colonies are brownish in the center and light brown
towards the edge (Fig. 4g). The colonies are light-brown
in the potato medium and white and wet in BDA (Fig. 4g).
It should also be noted that several new diazotrophic
Burkholderia species have been described that are symbi-
otic with legumes, most notably of Mimosa spp. in Brazil.
Some of these, such as B. phymatum and B. tuberum have
also been shown using semi-solid JMV medium plus
yeast extract to fix N2 ex planta (Elliott et al. 2007), which
is a highly unusual feature for legume-nodulating bacteria.
Additional media for enrichment and isolation
of N2-fixing bacteria
OAB semi-solid nitrogen-free medium (Okon et al.
1977)
The original NFb medium of Döbereiner and Day
(1976) was modified by Okon et al. (1977) to provide
Plant Soil (2014) 384:413–431
increased buffer capacity and micronutrient elements
to isolate and count Azospirillum lipoferum from
pure culture and from inoculated maize plants. The
medium contains the following (per liter of distilled
water): K2HPO4, 6.0 g; KH2PO4, 4.0 g (mixed in
0.1 the final volume and autoclaved separately from
the other medium constituents; the phosphate solu-
tion is later mixed with the cold medium);
MgSO4.7H20, 0.2 g; NaCl, 0.1 g; CaCl2, 0.02 g;
DL-malic acid, 5.0 g; NaOH, 3.0 g; FeCl3, 10.0 mg;
NaMoO4.2H20, 2.0 mg; MnSO4, 2.1 mg; H3BO3,
2.8 mg; CuSO4.5H2O, 0.04 mg and ZnS04.7H20,
0.24 mg. The final pH is adjusted to 6.8. For growth
on N2 under microaerophilic conditions, no NH4Cl
or yeast extract are added to the medium, but 0.5 g
of agar are added per liter.
Combined carbon or Rennie semi-solid medium
(Rennie 1981)
The combined carbon or Rennie semi-solid medium
(RM) is prepared from solutions A and B. Solution A
consists of 0.8 g of K2HPO4, 0.2 g of KH2PO4, 0.1 g of
NaCl, 28 mg of Na2FeEDTA, 25 mg of Na2MoO4.2H2O,
100 mg of yeast extract (Difco), 5.0 g of mannitol, 5.0 g
of sucrose, 0.5 ml of 60 % (vol/vol) sodium lactate and
900 mL of distilled water (the final pH of solution A is
adjusted to 7.0 before autoclaving). Solution B consists of
0.2 g of MgSO4.7H2O, 0.06 g of CaCl2.2H2O, and
100 mL of distilled water. The solutions are autoclaved
separately and mixed after cooling. Filter-sterilized biotin
and para-aminobenzoic acid (100 μl each) are added at
final concentrations of 5 and 10 μg L−1, respectively. Add
2.0 g and 15 g agar L−1 for semi-solid and solid medium,
respectively.
M medium (Xie and Yokota 2005)
The nitrogen-fixing bacterium Azospirillum oryzae (Xie
and Yokota 2005) was isolated using medium M which
contains (g L−1): sodium malate, 5.0; CaCl2.2H2O, 0.02;
MgSO4.7H2O, 0.2; K2HPO4, 0.1; KH2PO4, 0.4; NaCl,
0.1; FeCl3, 0.010; Na2MoO4.2H2O, 0.002; yeast extract,
0.1 and biotin (2 μg). Adjust the pH to 6.8 with KOH
and add distilled water to bring the total volume to 1,000
mL. To semi-solid medium add 2.0 g agar L− 1.
Azospirillum canadense (Mehnaz et al. 2007) can also
be isolated using M medium, except that biotin is omit-
ted and pH is adjusted to 7.2 to 7.4.
Plant Soil (2014) 384:413–431
Semi-solid synthetic malate (SSM) medium (Reinhold
et al. 1986, Reinhold et al. 1987)
The semi-solid synthetic malate (SSM) medium was
adapted for isolating diazotrophs from the high salt
concentrations commonly found in soils where Kallar
grass (Leptochloa fusca) is found. SSM was used for
enrichment and isolation of Azospirillum
halopraeferens and also of Azoarcus spp. (Reinhold-
Hurek et al. 1993). The medium has the following
composition (g L−1): malic acid, 5.0; KOH, 4.8; NaCl,
1.2; Na2SO4, 2.4; NaHCO3, 0.5; CaC12, 00.22; MgSO4
- 7H2O, 0.25; K2SO4, 0.17; Na2CO3, 0.09; Fe (II1)
(EDTA), 0.077; K 2 HPO 4 , 0.13; biotin, 0.0001;
MnC1 2. 4H 2 O, 0.0002; H 3 BO 3 , 0.0002; ZnC1 2 ,
0.00015; CuC12 - 2H2O, 0.00002; Na2MoO4 2H2O,
0.002 and 2 g agar. For solid medium add agar (8 g)
and yeast extract (0.1 to 0.2 g). The final pH of the
medium is adjusted to 8.5, and distilled water is added to
bring the total volume to 1 liter.
Media used for purification of diazotrophic bacteria
and stock solutions
BMS or Potato agar medium
BMS (Potato – malate – sucrose) medium has the fol-
lowing composition (g L−1): potatoes peeled and sliced,
200; DL-malic acid, 2.5; KOH, 2.0; crystalized cane
sugar, 2.5; vitamin solution (see above), 1.0 mL; Micro-
nutrient solution (see above), 2 mL; bromothymol blue
(5 g L−1 in 0.2 N KOH), 2 drops; agar, 15.0. The
potatoes are placed in a gauze bag, boiled in 0.5 l of
water for 30 min, and then filtered through cotton,
saving the filtrate. The malic acid is dissolved in
50 mL of water and the bromothymol blue added.
KOH is added until the malic solution is green
(pH 6.8-7.0). This solution, together with the crystalized
cane sugar, vitamins and agar, is added to the potato
filtrate. The final volume is made up to 1 L with distilled
water. The medium is boiled to dissolve the agar and
then sterilized by autoclaving.
Potato-P agar medium (Döbereiner 1995)
This is similar to the BMS medium, but the concentra-
tion of crystallized cane sugar is increased to 100 g L−1,
malic acid is omitted and the pH is adjusted to 5.5. This
425
medium is specifically applied to the final purification/
characterization of the N2-fixing Gluconacetobacter
diazotrophicus.
Rojo Congo (RC) agar medium (Rodriguez-Cáceres
1982)
The RC agar medium, claimed to improve the isolation/
identification of Azospirillum spp. (colonies show a
scarlet color), contains (g L−1): DL-malic acid, 5;
K2HPO4, 0.5; MgSO4.7H20, 0.2; NaCl, 0.1; yeast ex-
tract, 0.5; FeCl3.6H20, 0.015; KOH, 4.8; and agar, 20.
The pH is adjusted to 7.0 with 0.1 M KOH. A total of
15 mL of a 1:400 aqueous solution of Congo red
(autoclaved separately) is aseptically added to each liter
of the melted medium just before use.
DYGS medium – Rodrigues Neto et al. (1986) modified
DYGS medium was developed for the enrichment and
isolation of Xanthomonas spp. by Rodrigues Neto et al.
(1986). Later, the composition was modified and used
for the purification of diazotrophic bacteria. The com-
position is (g L−1): glucose, 2.0; malic acid 2.0; peptone,
1.5; yeast extract, 2.0; K2HPO4, 0,5; MgSO4.7H2O, 0.5;
glutamic acid, 1.5; complete with distilled water up to
1,000 mL. The optimum pH is 6.0 for Herbaspirillum
spp. and Gluconacetobacter spp. and 6.8 for
Azospirillum spp. Usually, Gluconacetobacter spp.
grow well using DYGS medium without malic acid.
Advantages and limitations of the N-free semi-solid
media
It is recognized by those working in the field of nitrogen
fixation with non-legumes that the development of the N-
free semi-solid medium allowed a tremendous accumula-
tion of knowledge on the ecology and physiology of
diazotrophic bacteria. At least 20 N2-fixing species have
been isolated and identified based on the enrichment
method originally developed by Döbereiner and Day
(1976). Because of the simplicity of the method, it has
also been used to estimate the population of diazotrophic
bacteria associated with rhizosphere soil and plant tissues,
although it almost certainly underestimates diazotrophic
bacterial populations as discussed previously. Depending
on the type of study, a specific culturable diazotrophic
species population may be counted, especially when
426
combining media with antibiotics to target specific
strains, as used in a study carried out by (Baldani
et al. 1986b, Baldani et al. 1987) to monitor the
establishment of inoculated Azospirillum brasilense
strains Sp245 str and Sp 107 str in wheat and
A. lipoferum strain Sp S82str in sorghum. In addition,
it is possible to perform ARAs directly on the bacteria
growing in the semi-solid medium or even to determine
their N2 fixation ability by measuring the 15 N incorpo-
rated into their cells after exposure of the culture to
15
N2 gas (Baldani et al. 1992). These procedures em-
phasize the importance of semi-solid media for wider
physiological characterization studies of diazotrophic
bacteria in programs designed to select strains for plant
inoculation or to exploit the diversity of N2-fixing
bacteria associated with various non-legumes.
Despite the aforementioned advantages of the semi-
solid medium, it presents some limitations that need to
be taken into account: a) The method allows the identi-
fication of only a fraction of the diazotrophic bacterial
population present in the plant tissues or in soils; b)
Although they were designed to be specific for particu-
lar diazotrophic genera, it is possible when using these
media to isolate other diazotrophic bacteria that use the
same carbon sources or which tolerate the same pH level
as for example the nitrogen-fixing species belonging to
the genera Stenotrophomonas, Pantoea, Enterobacter,
etc. that were isolated from sugarcane using the LGI,
LGI-P and JNFb media (Taulé et al. 2012); c) At least
100 bacterial cells are necessary to initiate growth and
the actual number of cells depends on the medium used
and, d) Non-N2-fixing bacteria (scavengers) can grow
together with the diazotrophs during the incubation
period (pellicle formation) due to the fixed nitrogen
released by the diazotrophic bacteria, and these may
interfere with the process of isolation and purification
of the target (diazotrophic) bacteria. In order to reduce
this latter problem, Döbereiner (1988) recommended
that the pellicle should be transferred to another semi-
solid medium before initiating the isolation process with
solid medium. It is important to verify the pellicle for-
mation, as many other (non-diazotrophic) bacteria can
utilize (i.e. scavenge) small amounts of fixed N present
in the medium. The “10 commandments” listed by
Döbereiner (1988) indicate the procedures, including
the necessity for the demonstration of least a minimal
degree of nitrogenase (acetylene reduction) activity, to
be followed when seeking success in isolating N2-fixing
bacteria from non-legumes.
Plant Soil (2014) 384:413–431
Future Perspectives
In conclusion, the introduction of the N-free semi-solid
medium opened new opportunities for those working in
the area of BNF with non-legumes, not only for the
important role played by microorganisms associated
with cereals and energy crops but also because some
of these diazotrophs, most notably Azospirillum spp.,
are being recommended as inoculants for sugarcane,
maize and wheat in Brazil and in other countries, such
as Argentina, Mexico, Colombia, Egypt and South Af-
rica, among others. Azospirillum spp. have also been
used as co-inoculant plant growth-promoting bacteria to
be applied with soybean rhizobia in Argentina and
South Africa for many years. The recommended meth-
odologies applied to count these bacteria in formulations
include recipes of semi-solid and solid media, thus
illustrating their importance in the general field of BNF.
Molecular approaches, such as qPCR can be applied
to detect with high efficiency a target N2-fixing species
or even a diazotrophic strain colonizing soil or plant
tissues (Quecine et al. 2012). nifH cDNA clone libraries
have also been applied to detect the functional
diazotrophic bacterial community associated with many
poaceous plants (Fischer et al. 2012; Videira et al. 2013;
Demba-Diallo et al. 2008; Ando et al. 2005). To facili-
tate the isolation of these in silico identified diazotrophic
bacteria, traditional microbiological methods (via isola-
tion using semi-solid media) can be coupled to a molec-
ular probe-directed approach that may be used to predict
the culturable diazotrophic diversity present in the plant
tissues (Hartmann et al. 2006). Several molecular probes
have already been designed (Stoffels et al., 2001;
Rothballer et al. 2006) and applied to monitor N2-fixing
proteobacteria associated with non-legumes (Schloter
et al. 1995; Oliveira et al. 2009).
The majority of the diazotrophic bacteria associated
with non-legumes have been obtained using the recipes
listed earlier. However, considering the published liter-
ature on this subject the possibilities to isolate new
species and even genera are very high using novel
semi-solid media. For example, a simple modification
in the NFb medium by the addition of 3 % NaCl allowed
the isolation from the Brazilian coral species
Mussismilia hispida of nineteen Vibrio strains belonging
to species V. harveyi, V. alginolyticus, V. campbellii, and
V. parahaemolyticus. Many of these vibrios were capa-
ble of growing six consecutive times in N-free medium
and each time showed strong nitrogenase activity
Plant Soil (2014) 384:413–431
(Chimetto et al., 2008). A similar strategy was used by
Jha et al. (2012). Simply by adding 4 % NaCl to the NFb
semi-solid medium enabled them to isolate many
diazotrophic Gram positive (Brachybacterium
saurashtrenese, Brevibacterium sp. and Zhihengliuella
sp.) and Gram negative (Haererehalobacter sp.,
Halomonas sp. and Mezorhizobium sp.) diazotrophic
bacteria from the halophyte Salicornia brachiata grow-
ing in India. The modification of the semi-solid LGI
medium by addition of chlorogenic acid, caffeic acid,
substituting glucose for sucrose and adjusting pH to 6.8,
allowed the isolation of an endophytic N2-fixing Kleb-
siella oxytoca strain from sweet potato (Ipomoea
batatas) stems growing in Japan (Adachi et al. 2002).
Other strategies to isolate new species, mainly those
identified in silico but not yet cultivated, should be
continuously investigated. For example, the use of a
small amount of filtered root or stem extract increases
the diversity of N2-fixing bacteria isolated from target
non-legumes, as exemplified by Elbeltagy et al. (2001)
who added rice shoot extracts to the semi-solid RM
medium (Rennie 1981) and this allowed for the isolation
from rice plants of several diazotrophic bacteria phylo-
genetically related to Herbaspirillum, Ideonella, Entero-
bacter, and Azospirillum. In this case, however, care
should be taken into considering the presence of the
nutrient N in the plant tissues used to supplement the
media, as it may stimulate the growth of non-N2-fixing
bacteria. The use of a semi-solid medium containing
more than one carbon source, mainly those related to
exudates released by the host plant, has also allowed for
the isolation of a greater diversity of N2-fixing bacteria.
For example, the use of semi-solid NFb COG medium
(malic acid replaced by citric acid, oxalic acid and
glucose - ratio 1:1:3 g L-l), pH 5.5, allowed for the
isolation of many diazotrophs from wetland rice plants,
including Azospirillum, Herbaspirillum and an uniden-
tified species named “Bacteria E” (Oliveira 1992),
which was later characterized as belonging to the genus
Burkholderia (Hartmann et al. 1995).
Therefore, the options for the creation of new semi-
solid media to survey new bacterial species and to
exploit the diversity of diazotrophic bacteria are vast
and depend only on the input of the younger research
scientists starting out in the field of non-legume BNF
that has just reached its half century. One example is the
successful strategy applied by Rouws et al. (2013) to
isolate many endophytic Bradyrhizobium strains from
sugarcane plants based on culture-independent
427
molecular information that indicated the presence of
“bradyrhizobia” colonizing the plant (Fischer et al.
2012; Burbano et al. 2011). Another example is the
approach carried out by (Lonhienne et al. 2014) that
first identified bacteria markedly enriched in the rhizo-
sphere of sugarcane plants based on culture-independent
bacterial community assessment using 16S rDNA
amplicon sequencing to guide the isolation of N2-fixing
strains, such as the new species Burkholderia australis
that was derived from their study.
Finally, in order to search for, isolate and confirm that
particular diazotrophs have an associative/endophytic
ability to colonize non-legumes and/or that they perform
efficiently when applied as a PGPR in agriculture, we
recommend a combination of sophisticated culture-
independent molecular approaches (including high res-
olution microscopy) together with the application of
simple microbiological methods that utilize standard
and/or modified nitrogen-free semi-solid media. Indeed,
the power of such a combined “traditional” and “molec-
ular” approach guarantees that it will most certainly be
required for many years to come.
Acknowledgments The authors thank Embrapa Agrobiologia,
the CNPq/INCT-FBN and FAPERJ for financial support and
CNPq for the fellowship of the researchers of Embrapa
Agrobiologia. Thanks also to our colleague Robert M. Boddey
for his encouragement to write this guide and reading the manu-
script. The authors thank the laboratory analist Fernanda Dourado
for the bacterial photographs.
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