Influence of nano nutrients on heterocyst-forming cyanobacterium Anabaena ambigua Rao

Article abstract of DOI:10.1080/15533174.2011.591875

The present study is deals with the influence of nanoparticles on the growth of heterocyst-forming cyanobacterium Anabaena ambigua Rao [A100]. The nano molybdenum and iron particles have been synthesized respectively from molybdenum trioxide (MoO₃) and iron chloride (FeCl₃) employing sodium borohydride (NaBH₄) as a reducing agent and characterized comprehensively. BG11 (N^-) media containing micronutrients like molybdenum and iron were replaced respectively by different concentrations of nano molybdenum [BG11 (N^-Mo^- + nano Mo)] (0, 5, 10, 15, 20, 25, 40, 60, 80, and 100%) and nano iron [BG11 (N ^-Fe^- + nano Fe)] (0, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 40, 60, 80, and 100%) particles for the cultivation. The effect of nano nutrients on cyanobacterial growth parameters like heterocyst formation, chlorophyll content, and protein content was analyzed. The obtained results showed that the high concentrations of nano molybdenum induce heterocyst formation, increase protein content, and decrease chlorophyll content when compared to the control, whereas the nano iron suppresses the growth of cyanobacteria Anabaena ambigua Rao [A 100]. Copyright Taylor & Francis Group, LLC.

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Influence of Nano Nutrients on Heterocyst-Forming
Cyanobacterium Anabaena ambigua Rao
R. Yuvakkumar a , V. Elango a , R. Venkatachalam a , N. Kannan b & P. Prabu a
a Centre for Nanoscience and Technology, K. S. Rangasamy College of Technology,
Tiruchengode, Tamilnadu, India
b Department of Biotechnology, K. S. Rangasamy College of Technology, Tiruchengode,
Tamilnadu, India
Published online: 02 Dec 2011.
To cite this article: R. Yuvakkumar , V. Elango , R. Venkatachalam , N. Kannan & P. Prabu (2011) Influence of Nano
Nutrients on Heterocyst-Forming Cyanobacterium Anabaena ambigua Rao , Synthesis and Reactivity in Inorganic, Metal-
Organic, and Nano-Metal Chemistry, 41:10, 1234-1239, DOI: 10.1080/15533174.2011.591875
To link to this article: http://dx.doi.org/10.1080/15533174.2011.591875
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Synthesis and Reactivity in Inorganic, Metal-Organic, and Nano-Metal Chemistry, 41:1234–1239, 2011
Copyright ꢀ Taylor & Francis Group, LLC
C
ISSN: 1553-3174 print / 1553-3182 online
DOI: 10.1080/15533174.2011.591875
Influence of Nano Nutrients on Heterocyst-Forming
Cyanobacterium Anabaena ambigua Rao
R. Yuvakkumar,1 V. Elango,1 R. Venkatachalam,1 N. Kannan,2 and P. Prabu1
1Centre for Nanoscience and Technology, K. S. Rangasamy College of Technology, Tiruchengode,
Tamilnadu, India
2Department of Biotechnology, K. S. Rangasamy College of Technology, Tiruchengode, Tamilnadu, India
nanoscale is more rapid than at macroscale. These nanostruc-
tured materials have become more significant and created an
impact in the areas of chemical, energy, electronic, and biologi-
cal sciences for wide range of applications.[2–4] The present fea-
sibility study focuses on novel macro- to nanoscale applications
on heterocyst-forming cyanobacterium Anabaena ambigua Rao
[A 100].
The growth of Anabaena depends on the availability of ni-
trogen in the form of amino acids, nucleic acids, proteins, etc.
In the atmosphere, nitrogen is present in the form of dinitrogen,
which is inert due to the triple bond. In order to utilize nitrogen
for the growth of cyanobacteria it must be “fixed” (combined)
in the form of ammonium or nitrate ions. All nitrogen-fixing
cyanobacteria require a nitrogenase enzyme complex, which
contains (Fe-Mo) cofactor. The nitrogen fixation occurs in many
strains of filamentous cyanobacteria under oxic growth condi-
tions in differentiated cells called heterocysts that maintain a
microxic environment to support nitrogenase activity. Hence,
the transition metals molybdenum and iron are the essential
micronutrients required for nitrogen metabolism in heterocyst-
forming cyanobacteria.[5–8]
The present study is deals with the influence of nanoparticles on
the growth of heterocyst-forming cyanobacterium Anabaena am-
bigua Rao [A100]. The nano molybdenum and iron particles have
been synthesized respectively from molybdenum trioxide (MoO3)
and iron chloride (FeCl3) employing sodium borohydride (NaBH4)
as a reducing agent and characterized comprehensively. BG11 (N−)
media containing micronutrients like molybdenum and iron were
replaced respectively by different concentrations of nano molyb-
denum [BG11 (N−Mo− + nano Mo)] (0, 5, 10, 15, 20, 25, 40, 60, 80,
and 100%) and nano iron [BG11 (N−Fe− + nano Fe)] (0, 0.1, 0.2,
0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 40, 60, 80, and 100%) particles
for the cultivation. The effect of nano nutrients on cyanobacterial
growth parameters like heterocyst formation, chlorophyll content,
and protein content was analyzed. The obtained results showed
that the high concentrations of nano molybdenum induce hetero-
cyst formation, increase protein content, and decrease chlorophyll
content when compared to the control, whereas the nano iron sup-
presses the growth of cyanobacteria Anabaena ambigua Rao [A
100].
Keywords chlorophyll, cyanobacteria, heterocyst, nano nutrients,
protein
Some filamentous cyanobacteria such as Nostoc punctiforme,
Cylindrospermum stagnale, and Anabaena sperica are able to
form nitrogen-fixing cells called heterocysts during nitrogen
starvation. They develop from vegetative cells and form char-
acteristic polar nodules and a thick cell wall. They fix nitrogen
from the air using nitrogenase enzyme, in order to provide the
required nitrogen to the cells for biosynthesis.[9] The heterocyst
creates a micro-anaerobic environment when oxygen is inac-
tivated. The heterocysts unique structure and physiology are
required for gene expression. Hence, there has been intense
interest in heterocyst-forming cyanobacterium Anabaena am-
bigua Rao [A 100] for the past two decades.[10–14]
INTRODUCTION
Due to the advancement of nanomaterials, there is an in-
creasing demand in the replacement of conventional materials
by nanostructured materials.[1] Nanostructured materials find
potential applications due to their high reactivity and higher
surface to volume ratio. The ratio of surface area to volume of
any material at the nanoscale improves the efficiency in nutrient
delivery, uptake, and utilization. The biochemical reaction at
In the present investigation, different concentrations of nano
Received 19 November 2010; accepted 13 May 2011.
The authors are very thankful to the Defense Research and De- molybdenum and nano iron particles have been supplemented
velopment Organization (DRDO), New Delhi for financial support to
into the cyanobacterial growth medium as nano nutrients instead
carry out this project (ERIP/ER/0705077/M/01/1015 dt. 20.11.07).
of adding micronutrients. These nano nutrients are expected to
Address correspondence to Rajendran Venkatachalam, Centre for
play a crucial role due to their increased reactivity, and hence
Nanoscience and Technology, K. S. Rangasamy College of Technol-
to influence the heterocyst counts instead of bulk materials. The
purpose of this research was to determine whether the media
ogy, Tiruchengode- 637 215, Tamilnadu, India. E-mail: veerajendran@
gmail.com
1234

INFLUENCE OF NANO NUTRIENTS
1235
variations of nano nutrients levels would make a difference in 11 medium was prepared in an Erlenmeyer flask by adding
cyanobacterial metabolism, by analyzing heterocysts frequency, conventional chemicals in deionized water; pH was adjusted to
chlorophyll content, and protein content. This is a novel ap- around 7.0.[15] The medium was kept in autoclave for steriliza-
proach to study the influence of nano nutrients in heterocyst tion and then allowed to cool. Then the collected cultures (10
production and thereby their effects in increasing nitrogen fixa- µL) were inoculated in BG11 medium and incubated at 298
tion.
2 K, at a light intensity of 1500 to 3000 lux, for a photoperiod
cycles of 13/11 h. This BG11 medium was used to maintain
cyanobacterial culture, which act as a starter culture.
EXPERIMENTAL METHODS
Preparation of Media and Inoculation
Synthesis of Nano Molybdenum
The effect of different concentrations of nanoparticles on the
growth of Anabaena ambigua Rao [A 100] was analyzed in
BG11 (N−), BG11 (N−Mo− + nano Mo), and BG11 (N−Fe− +
nano Fe) media.
The crystalline nano molybdenum particles were prepared
via the reduction of molybdenum trioxide with sodium borohy-
dride, following the reaction:
g
s
s
s
s
2
2MoO ( )+3NaBH ( ) → 2Mo( )+3NaBO ( )+6 ( ) [1]
BG11 (N −) medium
H2
3
4
BG11 (N−) medium was prepared by devoiding the nitro-
gen source of standard BG11 medium, in order to study the
filamentous and heterocystous cyanobacterium. The molybde-
num (0.0154 mg) and iron (0.1264 mg) requirements in 100
mL BG11 (N−) medium were provided in the form of sodium
molybdate (39 mg) and ferric ammonium citrate (120 mg). Then
the medium was kept for sterilization and allowed to cool. Then
10 µL inoculums of Anabaena were taken from starter culture
and inoculated in BG11 (N−) medium and incubated at 298
2 K, at a light intensity of 1500 to 3000 lux for a photope-
riod cycles of 13/11 h. This medium formulation was used as a
control.
The reactants molybdenum trioxide and sodium borohydride
were taken respectively in the molar ratio of 1:3 and pulver-
ized homogenously in a clean mortar at room temperature. The
mixture was transferred into a silica crucible and then placed
inside the muffle furnace under a nitrogen environment. The
temperature of the muffle furnace was increased from 310 K to
573 K with a continuous supply of nitrogen gas at a flow rate of
1500 mL min−1. The mixture was incubated for 2 h at the
same temperature under nitrogen environment. The produced
gases were let out and the muffle furnace was cooled down
to room temperature. Then the product mixture was passivated
with continuous purging of nitrogen gas. In order to obtain the
pure crystalline nano molybdenum, the mixture was suspended
in distilled water followed by thorough washing, filtering, ex-
changing with acetone for several times, and drying at room
temperature.
BG11 (N −Mo− + nano Mo) medium
BG11 (N−Mo− + nano Mo) medium was obtained by modi-
fying BG11 (N−) medium, wherein conventional sodium molyb-
date was replaced by nano molybdenum at different percentage
concentrations (0, 5, 10, 15, 20, 25, 40, 60, 80, and 100%) and
sterilized. Then 10 µL of Anabaena ambigua culture was inoc-
ulated in BG11 (N−Mo− + nano Mo) medium and incubated at
appropriate conditions as mentioned earlier.
Synthesis of Nano Iron
Iron nanoparticles were prepared in aqueous solution via the
reduction method by mixing equal volumes of 0.94 M NaBH4
and 0.18 M FeCl3, following the reaction
BG11 (N −Fe− + nano Fe) medium
2FeCl +6NaBH +18
H2O
→ 2Fe0+6NaCl+6 (OH) +21
B
H2
[2]
3
4
3
Similar to nano molybdenum medium preparation, BG11
(N−Fe− + nano Fe) medium was prepared by modifying BG11
(N−) medium, wherein conventional ferric ammonium citrate
was replaced by nano iron at different percentage concentrations
(0, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 40, 60,
80 and 100%) and then the culture was inoculated as mentioned
previously.
The borohydride solution was added drop by drop into iron
chloride solution with vigorous hand stirring. The borohydride
was added completely to accelerate the reduction reaction and
to obtain nano iron particles. At the end of the reaction, the black
nano iron particles were formed and then washed several times
with deionized water and ethanol. The nano iron particles were
obtained employing centrifugation at 4000 rpm. A thin layer
of ethanol was added to preserve the nano iron particles from
oxidation.
Growth Parameter Analysis
The effects of nano nutrients on the Anabaena growth pa-
rameters such as heterocyst frequency, chlorophyll content, and
protein content were analyzed.
Strains and Culture Conditions
Total heterocyst count
Anabaena ambigua Rao [A 100] cultures collected from Cen-
tre for Advanced Studies in Botany (CAS), Madras University,
The heterocysts formation of Anabaena sp. was observed
Chennai, India, were used in this experiment. The 100 mL BG under amicroscopeafter 10days of incubation. Thetotal number

1236
R. YUVAKKUMAR ET AL.
TABLE 1
of heterocyst was counted per 500 vegetative cells for all three
grown media.
Influence of nano molybdenum on cyanobacterial metabolism
Nano
Mo
(wt%)
Heterocyst
Chlorophyll estimation
frequency (per 500 Total chlorophyll Protein of
vegetative cells)
First, 1 mL of culture was collected from the grown medium
and centrifuged at 3000 rpm for 10 min. To the pellet, 4 mL
of 95% methanol was added and mixed well and then kept in
water bath at 333 K for 30 min. The loss of volume was made
up with methanol and the mixture was centrifuged at 3000 rpm
for 5 min. Then the optical density (OD) value was calculated
using a spectrophotometer at 650 and 665 nm against a methanol
blank.[16]
culture × 10−3
culture
Control
5
10
15
20
25
40
60
80
20
21
23
24
25
26
26
29
30
31
0.5665
1.0815
1.4950
1.0725
0.5195
0.7020
0.4485
0.4365
0.3855
0.4605
171.65
152.30
150.14
155.10
159.05
161.23
166.10
215.00
230.50
200.00
Protein estimation
First, 1 mL of culture was centrifuged at 3000 rpm for 10
min. Then 2 mL of phosphate buffer was added to the pellets,
homogenized, and centrifuged at 8000 rpm for 20 min to collect
supernatant. An equal volume of 10% trichloroacetic acid was
added to the supernatant and incubated at 310 K for 30 min.
The resultant solution was centrifuged at 8000 rpm for 20 min.
The pellet was dissolved in 5 mL of 0.1 N NaOH and mixed
well. Then the protein content was estimated using Lowry’s
method.[17]
100
in different concentrations was counted repeatedly up to 500
vegetative cells and tabulated (Table 1) along with control. The
results show that the inoculum from a culture grown in standard
BG11 (N−) medium contains an average of 20 heterocysts per
500 vegetative cells. On the other hand, the inoculum from a
culture grown in BG11 (N−Mo− + nano Mo) medium contains
an average of 26.1 heterocysts per 500 vegetative cells. The
increase in the concentration of nano molybdenum from 5 to
100% in BG11 (N−Mo− + nano Mo) medium increases the
heterocyst frequency from 21 to 31. Compared to control, the
higher induction in their vegetative growth was observed in all
nano-molybdenum-treated cultures. It may due to the cell wall
transporters, which uptake the nano molybdenum effectively. In
addition, molybdenum acts as a cofactor for the enzyme nitro-
genase for the fixation of nitrogen. This may be the reason for
the increase in the heterocyst frequency compared to control. A
similar result was obtained by using molybdenum and vanadium
for heterocyst differentiation.[19]
Table 1 shows the total chlorophyll content of Anabaena am-
bigua Rao [A 100] grown in different concentration of nano
molybdenum. The cultures added with 5, 10, and 15% con-
centration of nano molybdenum show an increase in the total
chlorophyll content when compared to the control. Further in-
crease in the concentration of nano molybdenum leads to a
decrease in the chlorophyll content. This reveals that a small
amount of nano molybdenum is favorable for the synthesis of
chlorophyll, whereas further increase in the concentration of
nano molybdenum degrades the photo system II that is induced
RESULTS AND DISCUSSION
Characterization of Nanoparticles
The diffraction peaks labeled as (110), (200), and (211) are in
good agreement with the Bragg peaks of molybdenum (Figure
1a). The obtained particle diameter manually examined from
transmission electron microscopy (TEM) images is around 100
nm (Figure 1b). The particle size distribution of nano molybde-
num is shown in Figure 1c.
The ultraviolet (UV)-visible spectrum of nano iron particle
is shown in Figure 1d. The two absorption peaks at wave-
lengths of 216 nm and 268 nm indicates the formation of
nano iron particles. The obtained particle diameter from TEM
images is around 40 nm (Figure 1e). The particle size dis-
tribution of nano iron is shown in Figure 1f. The obtained
nano molybdenum and nano iron particles respectively in the
range of 100 and 40 nm were used for the metal supplements
in BG11 medium.[18] The influence of nanoparticles on the
growth of cyanobacteria on different media has been carried out
further.
Effect of Nano Molybdenum on Anabaena ambigua Rao
[A 100]
The effects of nano molybdenum on heterocyst frequency, by nitrogenase, chlorophyllase, and other enzymes, and hence
chlorophyll content, and protein content were examined for as- there is inhibition of the biosynthesis of chlorophyll.[20]
sessing the algal responses. The growth of Anabaena ambigua
The estimated protein content of Anabaena ambigua Rao [A
[A 100] with different concentrations of nano molybdenum is 100] grown in different concentrations of nano molybdenum
shown in Figure 2a along with replicates. Figure 2b shows the has been tabulated (Table 1). The observed results reveal that
microscopic view of heterocyst development. After 10 days of there is no strong significant difference in the protein content
incubation, the heterocyst frequency of Anabaena sp. grown at lower concentration when compared with the control. An

INFLUENCE OF NANO NUTRIENTS
1237
FIG. 1. (a, b, c) XRD, TEM, and PSA studies of nano molybdenum. (d, e, f) UV, TEM, and PSA studies of nano iron.
increase in protein content has been observed at 60, 80, and protein content was increased when compared to the control.
100% of nano molybdenum concentration. As the concentra- This may be the reason for the increase in the total protein
tion of nano molybdenum increased, the protein content also content.
increased because there was an increase in the heterocyst fre-
In general, the changes occurred during the heterocyst forma-
quency. The heterocyst frequency requires nitrogenase, chloro- tion are: First, they produce three additional cell walls, including
phyllase, and other proteins for the growth. Hence, the total one of glycolipid that forms a hydrophobic barrier to oxygen;

1238
R. YUVAKKUMAR ET AL.
FIG. 2. (a) Cyanobacterial growth in BG11 (N−Mo− + nano Mo) medium containing different concentrations of nano molybdenum along with replicates. (b)
Heterocyst development (color figure available online).
second, there is the increase in production of nitrogenase and pared to the control. The initial Anabaena culture grown medium
other proteins involved in nitrogen fixation[20]; and third, there BG11 (N−Fe− + nano Fe) was first changed to yellowish green
is degradation of photo system II, which produces oxygen.
and then gradually became colorless. This discoloration sug-
Owing to the basic concepts already described, similar re- gested that the loss of pigmentation is due to the iron stress on
sults are obtained in the present study. The observed results are, the cell wall of Anabaena, which inhibits the cell from uptake of
first, that the increased heterocyst frequency due to nano molyb- nutrients. This may be due to a siderophore mechanism of iron
denum clearly reveals the production of additional cell walls; transport inside the Anabaena, which fails to uptake the metallic
second, that the increased total protein content with increase in iron. Similar reports showed that a siderophore-mediated iron
the concentration of nano molybdenum shows the production pathway may be involved in affecting all the cellular activities
of nitrogenase and other proteins involved in nitrogen fixation; related iron starvation. Moreover, at higher concentrations, it
and third, that the decreased chlorophyll content with increase in gives cytotoxic effects in various cellular processes like growth,
the concentration of nano molybdenum shows the degradation nitrogen fixation, CO2 fixation, and photosynthetic activities.[21]
of photo system II. Thus, it is clear from these observations that Hence, the cultures inoculated in BG11 (N−Fe− + nano Fe) me-
there is a positive influence of nano molybdenum on Anabaena dia are not able to grow after 10 days of incubation, as shown
ambigua.
in Figure 3.
On the other hand, the cultures grown in a lower concentra-
tion (<1%) of nano iron medium BG11 (N−Fe− + nano Fe) has
been regenerated when inoculated in BG 11 medium, as shown
in Figure 4. This may be due to the formation of akenites dur-
ing metal stress in Anabaena. The present study confirms that
the organisms undergo a dormant stage when they are grown in
a much lower concentration of nano-iron-supplemented BG11
Effect of Nano Iron on Anabaena ambigua Rao [A 100]
The effect of different concentrations of nano iron particles
on the growth of Anabaena was observed in BG11 (N−Fe− +
nano Fe) medium as shown in Figures 3a and 3b. After 10 days
of incubation, the growing culture loses its pigmentation with
increase in the concentration of nano iron from 1% when com-
FIG. 3. Cyanobacterial growth in BG11 (N−Fe− + nano Fe) medium: (a) lower concentration; (b) higher concentration (color figure available online).

INFLUENCE OF NANO NUTRIENTS
1239
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CONCLUSIONS
The influence of nano nutrients on the heterocyst-forming
cyanobacterium Anabaena sp. has been studied. A significant
increase in the heterocyst frequency of test organisms has been
achieved in BG11 (N−Mo− + nano Mo) medium. The cultures
applied with a low percentage (5, 10, and 15%) of nano molyb-
denum show an increase in total chlorophyll contents, while
the chlorophyll content decreases at higher concentrations (60,
80, and 100%) of nano molybdenum. The protein content is
low at low concentrations of nano molybdenum, whereas an
increase in the concentration of nano molybdenum increases
the protein content. In contrast, nano iron does not have any
strong significant effect on cyanobacterial metabolism. Thus,
the applications of nano nutrients play a significant role on the
heterocyst-forming cyanobacterium Anabaena ambigua Rao (A
100).
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