Diazotrophs are nitrogen fixing bacteria and carbohydrates sugars, sugar
alcohols, organic and amino acids are generally considered as carbon and
energy sources for the rhizospheric diazotrophs (Chan, 1986). Diazotrophs
utilized rhizosphere carbon substrates as their energy and fix nitrogen
form the atmosphere and form natural association with plants. About 64-86%
of the carbon released in to the rhizosphere is respired by microorganisms
(Hutsch et al., 2002). Diazotrophs activity is higher in the vicinity
of plant roots due to rhizospheric deposition. A 10-100 fold more microbial
population found in the rhizosphere as compared with the surrounding bulk
soil (Weller and Thomashow, 1994).
Carbon substrate utilization pattern are definite biochemical characteristics
of microorganisms. Diazotrophs shows differences in terms of minimal doubling
time, specific growth rate and consumed different amount of carbon substrates
as their energy sources. The carbon substrate utilization pattern also
differed when there is a mixture carbon compounds instead of sole carbon
sources. In mix carbon conditions bacteria often utilized one carbon source
preferentially and further carbon sources being consumed when the preferred
carbon source utilized (Wendisch et al., 2000). The cell generation
time depends on carbon substrates. This may vary from 10 min to several
days. Some of the uncultivated bacteria have longer doubling times (Couturier
and Rocha, 2006). Bacteria consumed carbon substrates and increases in
number following different growth model. The population growth increased
in specific substrates and reached in maximum number and after then decreased.
The metabolites produced in growth culture media inhibit further populations.
Diazotrophs mostly depends on rhizosphere carbon compounds. Carbon-substrate
availability is the most common limiting factor of microbial activity
in soil (Paterson et al., 2007). The aim of the present study
was to determine the sole carbon substrate utilization pattern of
diazotrophs and population growth on the specific carbon substrates. The
information can be used to apply specific carbon sources to support and
enhance a particular diazotrophs that can be used as a plant growth promoting
inoculants to maximize the potential of biological nitrogen fixation in
rice cultivation system.
MATERIALS AND METHODS
Burkholderia sp. (Sb13), Rhizobium sp. (Sb16) and two different
Corynebacterium spp. (Sb26 and Sb35) capable of high nitrogenases
enzyme activity and IAA production (previously isolated from rice root
environment) were tested for carbon substrate utilization in glucose,
fructose, sucrose and arabinose substrates. The carbohydrate sugars were
used as sole carbon source for bacterial growth. Sugar consumption, specific
growth rate and doubling time were determined. The experiment was laid
out in completely randomized design with 4 replications. Data were analyzed
using SAS (9.1version) statistical software.
Substrate preparation: Carbon and nitrogen free nutrient culture
solution was modified from Egener et al. (1999) with 15 g of individual
sugar (glucose, fructose, sucrose and arabinose) were added and used as
growth culture medium. Composition of the medium in 1 L were as follows:
KH2PO4, 1.5 g; K2HPO4, 0.33
g; K2SO4, 0.2 g; ferric citrate, 13 mg; CaCl2.2H2O,
0.4 g; MgCl2., 0.4 g; Na2MoO4.2H2O,
2 mg; H3BO3, 3 mg: MnSO4.H2O,
2 mg; ZnSO4.7H2O, 0.2 mg; CuSO4.5H2O,
Inoculum preparation: Four diazothrophic species were grown in
nutrient agar broth for 48 h and cells were harvested by centrifugation
at 13500 rev min-1 for 10 min in eppendorf tube and washed
with 0.85% sterilized Phosphate Buffer Saline (PBS) according to Bacteriological
Analytical Manual (2001). Composition of PBS (g L-1): 7.650
g NaCl, 0.724 g Na2HPO4 and 0.210 g KH2PO4.
After washes the bacterial cells were immediately suspended into PBS solution.
Optical density (OD 600) of washed cells were checked and adjusted
to 0.1. The population was confirmed by cell enumeration in drop plate
method on nutrient agar. Approximately 104 live bacterial cells
were applied to each sugar substrates. The inoculated conical flasks were
incubated at 28°C temperature on a rotary shaker.
Population growth determination: The bacterial cells were harvested
after 6, 12, 18, 24 and 36 h of incubation. Optical density was checked
by spectrophotometer at 600 nm wave length. 1 mL of culture solution from
each conical flask was diluted 10 fold up to 10-10 and 0.1
mL of each dilution was dropped on nutrient agar plates. Bacterial populations
were counted using drop plate count method.
Specific growth rate: Specific growth rate of each bacterial culture
at different sampling time was determined as:
where, μ is specific growth rate of individual culture. Value of
Z and Z0 represents the amount of any bacterial component of
the culture at times t and t0, respectively.
Doubling time: The mean doubling time or generation time were
where, g is time required for the culture to increase by a factor of
Sugar consumption: After every sampling, the bacterial culture
solution of each treatment were filtered through 0.2 μM pore syringe
filter and kept in -20°C temperature until analysis. A 20 μL
aliquot of samples were injected in high performance liquid chromatography
(HPLC, model Jasco Brownian). The amount of residual glucose, fructose,
sucrose and arabinose, were determined using Apex column (60°C) and
Refractive Index (RI) detector. Acetonitrile (75%) was used as mobile
phase with a flow rate of 1.8 mL min-1. A standard series of
0.25, 0.50, 0.75 and 1% sugars were prepared and sugar concentrations
were determined using standard graph. The amounts of sugar consumption
were determined as:
where, C is Amount of substrate consumption, Sto represent
substrate added at initial time and St is the substrate remaining
in the culture solution at each sampling time.
Burkholderia sp. (Sb13): Diazotrophic populations showed
a large variance in terms of population growth, generation time and followed
different specific growth rate models. The Burkholderia sp. (Sb13)
produced maximum population within 6 to 18 h of incubation period in glucose,
fructose, sucrose and followed exponential model of specific growth rate
(Fig. 2a-c). In glucose Burkholderia sp. obtained
maximum population growth at 6 h of incubation period and after then population
gradually decreased (Fig. 1). The cell generation
time was 0.41 h and consumed 97% of glucose within 36 h (Table
1) of incubation period. In fructose Burkholderia sp. obtained
maximum population growth at 12 h. The cell generation time 0.55 h and
after 36 h of incubation period consumed 96% of applied fructose. The
maximum population obtained at 18 h in sucrose. The doubling time 1 h
and consumed 96% of applied sucrose. In arabinose Burkholderia sp.
showed a gradually increased of population growth and at 36 h obtained
maximum population. The cell generation time was 1.2 h and specific growth
rate followed logarithmic model (Fig. 2d). After 36
h of incubation period 100% of arabinose was consumed by the Burkholderia
||Comparison growth of different strains in (a) glucose,
(b) fructose, (c) sucrose and (d) arabinose substrate. Values are
means of 4 replications. Error bars are standard errors of 4 replications,
Sb13; Burkholderia sp., Sb16; Rhizobium sp., Sb26; Corynebacterium
sp., Sb35; Corynebacterium sp. and Burkholderia sp.
||Specific growth rate (h) of Burkholderia sp.
(Sb13) in (a) glucose, (b) fructose, (c) sucrose and (d) arabinose
||Cell generation time and percent sugar consumption of
diazotrophs in different carbon substrates (values were means of 4
||Specific growth rate (h) of Rhizobium sp.(Sb16)
in (a) glucose, (b) fructose, (c) sucrose and (d) arabinose substrates
Rhizobium sp. (Sb16): The Rhizobium sp. population
gradually increased in glucose substrate and maximum population obtained
at 36 h (Fig. 1) of incubation period. The cell doubling
time 2.5 h and specific growth rate followed logarithmic model of growth
phase (Table 1, Fig. 3a). At 36
h of incubation period Rhizobium sp. consumed 90% of applied glucose.
In the fructose, Rhizobium sp. showed comparatively faster growth
rate and followed exponential model (Fig. 3b). The
maximum population obtained at 12 h and after then population decreased.
The cell generation time 0.62 h and consumed 94% of applied fructose (Table
1). The Rhizobium sp. obtained maximum population growth at
18 h of incubation period in sucrose and followed exponential model for
specific growth rate (Fig. 3c). The cell doubling
time 1 h and after 36 h of incubation period it consumed 80% of applied
sucrose. In arabinose substrate, Rhizobium sp. showed polynomial
model of growth phase (Fig. 3d) and generation time
1.1 h. It consumed 98% of arabinose after 36 h of incubation period.
Corynebacterium sp. (Sb 26): The population growth of Corynebacterium
sp. (Sb26) in glucose substrate gradually increased and maximum population
obtained at 36 h of incubation period following logarithmic model of specific
growth rate (Fig. 1, 4a). The
cell doubling time 1.86 h and consumed 96% of applied glucose. In the
fructose Sb26 showed faster growth rate and maximum population obtained
at 12 h. The specific growth rate in fructose followed exponential model.
The cell generation time 0.58 h and consumed 93.33% of applied fructose.
In the sucrose Sb26 produced maximum population at 24 h of incubation
period. For specific growth rate it followed power model. The generation
time 1.26 h and consumed 69.33% of applied sucrose. In arabinose, Sb26
obtained maximum population at 24 h and followed logarithmic model for
specific growth rate. The cell generation time 0.68 h and it consumed
100% of applied arabinose (Table 1, Fig.
||Specific growth rate (h) of Corynebacterium sp.
(Sb26) in (a) glucose, (b) fructose, (c) sucrose and (d) arabinose
||Specific growth rate (h) of Corynebacterium sp.(Sb26)
in (a) glucose, (b) fructose, (c) sucrose and (d) arabinose substrates
Corynebacterium sp. (Sb35): The specific growth rate of
Corynebacterium sp. (Sb 35) followed logarithmic model in four
different carbon substrates (Fig. 5a-d). In glucose
substrate population increased gradually and reached maximum at 36 h of
incubation period (Fig. 1). The cell generation time
2.06 h and consumed 94.64% of applied glucose (Table 1).
In the fructose substrate the maximum population obtained at 24 h of incubation
period (Fig. 1). The generation time 1.27 h and consumed
98% of applied fructose (Table 1). In the sucrose substrate
the maximum population obtained at 24 h and generation time 1.01 h. After
36 h of incubation period Sb35 consumed 94.67% of applied sucrose. In
arabinose the generation time 1.2 h and consumed 93.33% of applied arabinose.
The population of four diazotrophs showed a large variation in growth
rate and generation time in different carbon substrates. Generally eukaryotes
followed logarithmic growth model of growth phase. But the specific growth
rate highly depended on the substrates and presence of nutrient elements.
The generation time of diazotrophs even faster in the presence of nitrogen
element in the growth culture media (Chan, 1986). The generation time
also depends on substrate affinity. The Burkholderia sp. (Sb13)
showed high affinity to glucose and fructose and followed exponential
model of growth phase. The cell generation in four carbon substrates were
glucose > fructose > sucrose > arabinose. Although the specific
growth rate and cell generation was faster in other three substrates but
arabinose consumption was higher which indicates the gradual increased
in population which followed logarithmic model of growth phase maintained
living cells for longer time. The exponential growth phase cause early
maximum population growth and further population growth limited by accumulation
of inhibitory metabolites or end products. Limitation of biological space
and available nutrients also caused rapid death of cells. At the death
phase, the number of viable cells decreased exponentially, the reverse
growth of log phase (http://www.textbookofbacteriology.net/).
Rhizobium spp. (Sb16) grows rapidly in glucose, arabinose, fructose
and sucrose substrates (Vincent et al., 1979). In the present
study Sb16 showed high affinity to fructose compared to glucose. The cell
generation time were fructose > sucrose > arabinose > glucose.
Neal and Walker (1935) found superior growth of some Rhizobium
spp. in arabinose and other pentose sugar compared to glucose. Sb16 showed
logarithmic model of growth phase in glucose where as in fructose and
sucrose it showed exponential growth phase. Among four substrates sucrose
consumption is lower this may be due to the rapid breakdown of sucrose
in the solution and formed glucose and fructose. Sucrose consumption may
not reflect the actual consumption and generation time of microbes in
that particular disaccharide sugar.
Corynebacterium spp. (Sb26 and Sb35) showed a large variation
in population growth in different substrates. The generation time of Sb26
was fructose > arabinose > sucrose > glucose. Sb26 followed logarithmic
model of growth phase in glucose and arabinose. While in fructose it followed
exponential model for specific growth rate. Girbal et al. (2000)
recorded accelerating growth of C glutanimicum in fructose substrate
instead of a constant growth rate on glucose. Seibold et al. (2007)
also found early exponential growth of Corynebacterium glutamicum
in fructose substrate. The higher arabinose consumption may be due to
the gradual increased in population growth which produced less inhibitory
metabolites and maintained longer stationary phase of population growth.
The Corynebacterium sp. (Sb35) showed prolonged growth period in
four carbon substrates compared to other tested diazotrophs. The cell
generation time in different substrates were sucrose > arabinose >
fructose > glucose. Although the substrate affinity is different but
followed logarithmic model of growth phase and consumed almost similar
amount of carbon substrates.
The faster generation time and amount of substrate consumption not followed
the same pattern. The exponential growth phase doesn`t longer time. The
inhibitory product cause rapid cell death in closed culture system. The
cell division in a constant rate only depends upon the composition of
the growth medium and the conditions of incubation. In the stationary
phase viable cells can be determined but some of them may be stopped further
generation. Sb16 and Sb26 consumed lower amount of sucrose. The sucrose
consumption concealed with glucose and fructose and in mix carbon compound
bacteria showed different consumption as well as growth rate than sole
carbon source. In the sucrose the cell generation time was ± 1.0
for all four diazotrophs.
The diazotrophs exhibited differences in the specific growth rate, generation
time and utilized sugar as energy and carbon sources. Burkholderia,
Rhizobium and Corynebacterium spp. (Sb26) showed rapid growth
in fructose and followed exponential growth model for specific growth
rate. From the above study we found that logarithmic growth model sugar
consumption were high compared to other exponential, power and polynomial
growth model. Burkholderia sp. showed shorter generation time and
the Corynebacteria sp. (Sb35) showed longer generation time (>1.0
h) in all four carbon substrates. The application of glucose in Burkholderia
sp. (Sb13), fructose in Rhizobium sp. (Sb16) and Corynebacteriaum
sp. (Sb26) and sucrose in Corynebacteriaum sp. (Sb35) may accelerate
the growth of these diazotrophs in rice production system.
The author is grateful to the Universiti Putra Malaysia and Third World
Organization for Women in Science (TWOWS) for providing the financial
supports for the Ph.D project.