FIELD OF THE INVENTION
The present invention relates to a biofertilizer and its process of production thereof
comprising aerobic microbial consortium comprising of microbes of Escherichia coli DHl and
Pseudomonas sp BWDY-42 favouring mobilization of nitrate into nitrogen and plant growth
promotion. The present invention, more particularly, relates to the said biofertilizer
favouring nitrate detoxification in soil thereby converting it to nitrogen and facilitating
uptake by plants through nitrogen fixation to finally aid in plant growth promotion with
regard to increased shoot length, number of nodes, inter node length and number of leaves.
Advantageously, the said biofertilizers include fertilizers for agriculture and gardening that is
free of excess nitrate adapted for existing soil nitrate remediation and mobilization into
nitrogen to aid uptake by plants through nitrogen fixation. More advantageously, the said
biofertilizers facilitating soil nitrate reduction is poised to provide for a dual effect of plant
growth promotion and as well ensures that the agricultural runoff, if provided appropriate
time of incubation with plants, would not pollute the surrounding aquatic body.
BACKGROUND ART
Farmers use fertilizers to crops so that they grow better. But these fertilizers are generally
washed through the soil by rain, to end up in rivers. If large amounts of fertilizers or farm
waste drain into a river the concentration of nitrate and phosphate in the water increases
considerably. Algae use these substances to grow and multiply rapidly turning the water
green. This massive growth of algae, called eutrophication, leads to pollution. When the
algae die they are broken down by the action of the bacteria which quickly multiply, using
up all the oxygen in the water which leads to the death of many animals.
With the ever growing problem of population expansion and decreasing cultivable land for
food production, the use of nitrogen-rich fertilizer was almost inevitable. This has led to the
leaching of Nitrate to surface as well as ground water bodies. While nitrate is a common
nitrogenous compound due to the natural process of the nitrogen cycle, there are some
anthropogenic sources which enhance the nitrate concentration, particularly in the ground
water. These include fertilizers, cultivation, waste disposal, geologic reasons, precipitation,
irrigation and radioactive waste. Irregular rainfall and stream flow patterns, traditional
agricultural practices like dry farming, marginal irrigation, large scale flood plain farming,
application of fertilizers are considered as diffused sources of nitrate in soil and aquifers
(Whitmore, A.P., Bradbury, N.J., Johnson, P.A., Agriculture Ecosystems & Environment,

1992; Jorgensen, L.A., Water Science and Technology, 1999; Young, L., Zhamng, J., Water
Science and Technology, 1999; Burkart, M.R., Kolpin, D.W., James, D.E., Water Science and
Technology, 1999; Giupponi, C, Rosato, P., Water Science and Technology, 1999; Agrawal,
G.D., Water Science and Technology, 1999). Nitrate pollution is reported from paddy as well
as tea cultivation where the nitrate leaching causes acidification of soil and increases the
intensity of nitrate level in ground water (Nakasone, H., Yamamoto, T., Water Environ,
2004; Kinoshita, T., Katoh, T., Tsuji, M., Kanada, A., Inoue, T. Japanese Journal of Soil
Science and Plant Nutrition). The reuse of agricultural land for settlement causes
disturbance of soil layer during house building and leads to increase in nitrate level (Wakida,
F.T., Lerner, D.N., Water Research, 2005; Wakida, F.T., Lerner, D.N., Hydrological
Processes, 2006).
Nitrate is not a new problem and a health hazard. Excessive concentrations were recorded
in many domestic wells in a survey conducted 100 years ago. What is new is the public
concern about nitrate. This arises from two medical conditions that have been linked to
nitrate: methaemoglobinaemia ('blue-baby syndrome') in infants, and stomach cancer in
adults.
There are reports of leaching of nitrate from tea cultivation reducing the pH of the soil and
the runoff water. The use of No3 fertilizer is essential for the growth of the shoot as well as
the quality of the tea (especially in case of green tea). But only 12% (Yamada, T. Journal of
Water and Environment Technology, 2009) to 30% of the nitrate from the fertilizer (mostly
ammonium sulphate) is utilized by the plant while the rest adds to the pollution load. It
increases the acidity of the soil which in turn causes leaching of nutrients from the soil, and
accumulation of Aluminium. When this acidic water drains into the surrounding water bodies
with the aluminium and the nutrients, it causes death of the aquatic fauna (Nakasone, H.
and Yamamoto, T. Paddy Water Environ. 2004; Kinoshita, T. Japanese Journal of Soil
Science and Plant Nutrition; Kamiya, S. and Satake, K. http://es.ris.ac.jp/~satake/ Nitrate
pollution and acidification of the tea field located at Southern part of Shizuoka prefecture).
Nitrate pollution is a global problem and happens to be the second most dangerous
pollutant after the pesticides. When it leaches into the marine environment, it triggers
plankton bloom which in turn results in increased BOD in those areas. This process
decreases the available oxygen and thus kills the preexisting flora and fauna in those
regions (Thorburn et al. 2003 Agric Ecosys Environ 94:49-58).
Nitrate reducing bacteria constituting of a wide group of aerobes as well as certain group of
anaerobes are reported to participate in the denitrification phenomenon in the nitrogen

cycle. Under both organic and inorganic farming, nitrate in some form is added to the soil.
In case of biofertilizer application, microbes for nitrogen fixation are added to the soil or the
seed to enhance their nitrogen fixing ability. But none of these approaches teaches
utilizing/mobilizing the soil nitrate for plant growth.
Nitrate contamination is a potential human health threat especially to infants, causing the
condition known as methemoglobinemia, also called "blue baby syndrome. Central Nervous
System and Cardiovascular Vascular System may also be effected while it posses to be
carcinogenic (Clifford and Liu 1993 Amer Water Works Assoc J 85:135-143).
There are patents on formulation for nodulation and nitrogen fixation in legumes (US
2005/0187107 Al; US 4,878,936; US 5041383); on plant growth promotion (US 5503651,
US5503652, US6524998 Bl, US6855536 B2); on use of Lipo chitooligosaccharide
compounds for inducing early flowering in leguminous and non leguminous plants
(US2007/0027032 Al).
As apparent from the abovesaid, the addition of fertilizer, be it organic or inorganic, for
plant growth promotion apart from the other is both a common practice as well as a major
problem in terms of addition of excessive nitrate to the soil and also into the
River/lakes/aquifer through agricultural runoff causing major health problems.
Thus avoiding nitrate addition to soil in the form of fertilizer results in compromising with
plant growth. Thus maintaining the environmental health along with sustained plant growth
is a major challenge.
Also as discussed above for biofertilizer application, microbes for nitrogen fixation are added
to the soil or the seed to enhance their nitrogen fixing ability, and therefore, none of the
above approaches teaches utilizing/mobilizing the soil nitrate for plant growth.
Hence, it is imperative to provide for a biofertilizer which would have the dual effect of both
soil nitrate mobilization and plant growth promotion, which would itself be free from the
externally added nitrates thereby facilitating the conversion of the soil nitrate into nitrogen
to be picked up by the plants through nitrogen fixation process to induce branching as well
as nodulation of root in the plants for entrapping the released nitrogen source.
OBJECTS OF THE INVENTION
It is thus the primary object of the present invention to provide for a biofertilizer and its
process of production thereof comprising aerobic microbial consortium that would favour
mobilization of soil nitrate into nitrogen and plant growth promotion.
It is another object of the present invention to provide for a biofertilizer comprising aerobic
microbial consortium free from the external nitrate source which would perform a dual
function of leaching the nitrate from the soil and converting it to nitrogen through nitrogen
fixation.
It is yet another object of the present invention to provide for the said biofertilizer
comprising aerobic microbial consortium favouring dual function of nitrate detoxification in
soil and plant growth promotion in terms of shoot length, number of nodes; inter node
length and number of leaves.
It is still another object of the present invention to provide for the said biofertilizer that
would substantially decrease the need of adding external source of nitrate and instead
would facilitate mobilization of the excess nitrate of the soil.
It is also an object of the present invention to provide for the said microbial consortium that
can be added to any conventional fertilizer to thereby reduce the effective quantity of
fertilizer such that the excess amount of existing nitrates in the soil can be used as part of
remediation.
It is yet another object of the present invention to provide for the said biofertilizer such that
the microbial profile of the epiphytic leaf surface obtained on employing the said biofertilizer
for its cultivation did not show any increase at the qualitative level to reflect any adverse
affect.
It is still another object of the present invention to provide for the said biofertilizer
comprising the said microbial consortia which is not virulent for humans or livestock.
SUMMARY OF THE INVENTION
Thus according to the basic aspect of the present invention there is provided a biofertilizer
comprising aerobic microbial consortium comprising of microbes of Escherichia coli DHl and
Pseudomonas sp BWDY-42 and/or which have substantial identity with said Escherichia coli
DHl and Pseudomonas sp BWDY-42 favouring mobilization of nitrate into nitrogen and plant
growth promotion.
According to a preferred aspect of the present invention there is provided the said
biofertilizer wherein said microbial consortium is obtained from municipal sewage through
enrichment in nitrate broth.
In the backdrop of the related conventional biofertilizers comprising microbes for nitrogen
fixation or enhancing the nitrogen fixation ability of the soil, it comes as a surprising finding
by way of the present invention in providing a biofertilizer involving the said aerobic
microbial consortium comprising of selective microbes of Escherichia coli DHl and
Pseudomonas sp BWDY-42 and/or which have substantial identity with said Escherichia coli
DHl and Pseudomonas sp BWDY-42 which is found to surprisingly enable nitrate
detoxification in soil by way of mobilization of soil nitrate into nitrogen and plant growth
promotion thereby playing a dual role, which thus constitutes a significant technical
advancement in the related art.
According to another preferred aspect of the present invention there is provided the said
biofertilizer wherein said aerobic microbial consortium comprises of said microbes
Escherichia coli DHl and Pseudomonas sp BWDY-42 and /or microbes having said
substantial identity with said Escherichia coli DHl and Pseudomonas sp BWDY-42 in the
proportion of about 82:18.
According to yet another preferred aspect of the present invention there is provided the said
biofertilizer wherein said microbes having substantial identity with said Escherichia coli DHl
and Pseudomonas sp BWDY-42 comprises with about 97-99% identity at the partial 16S
rDNA level with the said microbes and non-virulent to humans or livestocks.
According to another preferred aspect of the present invention there is provided the said
biofertilizer wherein said aerobic microbial consortium is adapted for mobilization of nitrate
into nitrogen including reduction of nitrate in the solution with the nitrate concentration at
<5000ppm and >500ppm within 4 hours; and leaching nitrate from soil while passing
through to thereby favour soil nitrate detoxification and plant growth promotion.
According to another preferred aspect of the present invention there is provided the said
biofertilizer comprising said aerobic microbial consortium adapted for plant growth
promotion by inducing branching as well as nodulation of root for entrapping the released
nitrogen source and preponing the initiation of flowering and podding.
According to another aspect of the present invention there is provided the said process for
producing the biofertilizer comprising:
selectively providing an aerobic microbial consortium comprising of microbes of Escherichia
coli DHl and Pseudomonas sp BWDY-42 and/or which have substantial identity with said
Escherichia coli DHl and Pseudomonas sp BWDY-42 favouring mobilization of nitrate, into
nitrogen and plant growth promotion.
According to a preferred aspect of the present invention there is provided the said process
for producing the biofertilizer wherein said selectively providing of said aerobic microbial
consortium comprising of microbes comprises obtaining the same from municipal sewage
through enrichment in nitrate broth and with said Escherichia coli DHl and
Pseudomonas sp BWDY-42 and/or microbes having substantial identity with said Escherichia
coli DHl and Pseudomonas sp BWDY-42 in the proportion of about 82:18.
According to a yet another preferred aspect of the present invention there is provided the
said process for producing the biofertilizer comprising step of isolation of said aerobic
microbial consortium involving the steps of
a. acquiring municipal sewage followed by addition of a proportion (1%) of the same to
nitrate broth;
b. repeated cultivation at an optimum temperature preferably at 37 °C; and
c. obtaining therefrom the said aerobic microbial consortium through enrichment.
The objects and advantages of the present invention are described in greater details with
reference to the following non limiting accompanying examples, tables and figures.
BRIEF DESCRIPTION OF FIGURES:
Fig. 1: illustration of plant growth promotion by the biofertilizer comprising the aerobic
microbial consortium.
DETAILED DESCRIPTION OF THE INVENTION
As discussed hereinbefore, the present invention thus provides for a biofertilizer and its
process of production thereof comprising aerobic microbial consortium comprising of
microbes of Escherichia coli DHl and Pseudomonas sp BWDY-42 favouring mobilization of
nitrate into nitrogen and plant growth promotion. The said mobilization of the nitrate is
related to the nitrate mobilization of the soil thereby transforming it to nitrogen which in
turn is picked up by the plants by the process of nitrogen fixation ensuring increase in plant
length. Thus the said biofertilizer performs the dual function of nitrate detoxification in soil
and plant growth promotion in terms of shoot length, number of nodes; inter node length
and number of leaves.
This consortium is of immense importance for leafy vegetables as it increases the number of
leaves in treated plants.
Several plants were tested and the possible mechanism for plant growth promotion in Vigna
radiata might be through enhancement of nodulation. The epiphytic microbial population
(leaf surface) upon treatment does not show any increase in microbial variety at the
qualitative level indicating no negative effect of the applied microbial consortia. The
flowering in turn was early in case of microbe treated plants.
Example I: Microbial Consortia and its process of isolation
The microbial consortium was obtained from the municipal sewage through enrichment in
nitrate broth. It was stabilized through repeated cultivation at 37°C before being
characterized. This consortium is a mixture of microbes showing about 97-99% identity at
the partial 16S rDNA level with microbes like Escherichia coli DHl and
Pseudomonas sp BWDY-42 in the proportion of 82:18. It would reduce higher concentration
of nitrate (5000ppm< but >500ppm) from solution within 4hours while they could leachout
nitrate from soil while passing through it. This property of leaching would ensure making the
nitrate available to the plants while the reduction ensures that the agricultural runoff if
provided appropriate time of incubation would not pollute the surrounding aquatic body.
Example II: Method of testing nitrate reduction and plant growth
Seeds of Cicer arietinum and Vigna radiate were soaked on moist cotton and sprouting was
obtained. On the third day the sprouts were planted in small pots. On the seventh day of
sprouting they were transferred from small pots to test tubes with one plant in each tube
each. Before sowing, the plants were used for monitoring the height. After sowing, 500^1 of
the culture was added to the soil around the stem in treated set and same volume of nitrate
broth in case of control. It was allowed to grow for 20 days before being harvested for
measurement of Plant growth promotion (Fig. 1).
The remaining plants from the smaller pot were shifted to larger pots on the 10th day from
soaking. The plants were allowed to stabilize in the pots for 4 days before confluent culture
(50ml of confluent culture for 6 plants) was added on the 14th day and the plants were
allowed to grow till 40th day before they were harvested for measuring plant growth
promotion as compared to the initial data taken during planting in the large pots. Another
set was planted simultaneously and after 8 days of stabilization, the consortium was
inoculated in the soil. After 13 days and on the 14th day the leaf surface epiphytic
population was checked and showed no difference at the qualitative level as compared to
untreated plant indicating no adverse effect. The treated plants showed flowering within 46
days where as no such phenomenon was observed in case of control set. The data (provided
below) clearly shows an increase in shoot length, number of leaves and number of nodes
upon treatment in case of Cicer arietinum.
Tablel: Plant growth parameters of Cicer arietinum as a result of microbial treatment.
There was a significant increase in the number of nodules in case of Vigna radiata along
with increased root branching.
Table2: Plant growth parameter of Vigna radiata as a result of microbial treatment.
The epiphytic leaf surface microbial profile did not show any increase at the qualitative level
to reflect any adverse affect of the consortium application on the epiphytic profile.
It is thus possible for the said consortium included in the biofertilizer to prepone the
initiation of flowering and podding in case of Vigna radiata. The experiments were also
carried out in soil directly with similar results.
Therefore, the biofertilizer including the said consortium of the present invention would
substantially decrease the need of adding nitrate from outside and instead the excess
nitrate in soil would be mobilized for the purpose. Further the said present invention
ensures the provision of adding the said microbial consortia to conventional fertilizers to
favour reduction in the quantity of fertilizers required to promote plant growth wherein the
excess amount of existing nitrates of the soil can be used up as part of remediation. The
biofertilizer would thus find its end use and application in agriculture, gardening especially
coupled to environmental utilization of nitrate and would facilitate cultivation of legumes
and salad leaves / green leafy vegetables.
The advantages derived out of the biofertilizer of the present invention are outlined in
greater detail pointwise:
a. The soil would be treated for nitrate over load and the nitrate in turn would be
converted to nitrogen which would be used by the plant through nitrogen fixation;
b. The nitrate from soil would be mobilized for use as fertilizer instead of addition of
external sources of nitrate;
c. The epiphytic leaf surface microbial profile did not show any increase at the
qualitative level to reflect any adverse affect of the consortium application on the
epiphytic profile;
d. The microbial consortia is not virulent for humans or livestock;
e. It would have immense application in tea cultivation as nitrate is essential for green
tree quality yet only 12 to 30% of the nitrate is utilized while the rest is washed off
causing major problem in downstream water bodies.
Thus the technical advance made by way of the present invention is in providing a
biofertilizer comprising the said aerobic microbial consortium as a source of nitrogen to the
plants where the microbes perform dual function of leaching nitrate from the soil as well as
converting it to nitrogen and inducing branching as well as nodulation of root for entrapping
the released nitrogen source.
I Claim:
1. A biofertilizer comprising aerobic microbial consortium comprising of microbes of
Escherichia coli DHl and Pseudomonas sp BWDY-42 and/or which have substantial identity
with said Escherichia coli DHl and Pseudomonas sp BWDY-42 favouring mobilization of
nitrate into nitrogen and plant growth promotion.
2. A biofertilizer as claimed in claim 1 wherein said microbial consortium is obtained from
municipal sewage through enrichment in nitrate broth.
3. A biofertilizer as claimed in anyone of claims 1 or 2 wherein said aerobic microbial
consortium comprises of said microbes Escherichia coli DHl and Pseudomonas sp BWDY-42
and /or microbes having said substantial identity with said Escherichia coli DHl and
Pseudomonas sp BWDY-42 in the proportion of about 82:18.
4. A biofertilizer as claimed in anyone of claims 1 to 3 wherein said microbes having
substantial identity with said Escherichia coli DHl and Pseudomonas sp BWDY-42
comprises with about 97-99% identity at the partial 16S rDNA level with the said microbes
and non-virulent to humans or livestocks.
5. A biofertilizer as claimed in anyone of claims 1 to 4 wherein said aerobic microbial
consortium is adapted for mobilization of nitrate into nitrogen including reduction of nitrate
in the solution with the nitrate concentration at <5000ppm and >500ppm within 4 hours;
and leaching nitrate from soil while passing through to thereby favour soil nitrate
detoxification and plant growth promotion.
6. A biofertilizer as claimed in anyone of claims 1 to 5 comprising said aerobic microbial
consortium adapted for plant growth promotion by inducing branching as well as nodulation
of root for entrapping the released nitrogen source and preponing the initiation of flowering
and podding.
7. A process for producing the biofertilizer as claimed in anyone of claims 1 to 6 comprising:

selectively providing an aerobic microbial consortium comprising of microbes of Escherichia
coli DHl and Pseudomonas sp BWDY-42 and/or which have substantial identity with said
Escherichia coli DHl and Pseudomonas sp BWDY-42 favouring mobilization of nitrate into
nitrogen and plant growth promotion.
8. A process for producing the biofertilizer as claimed in claim 7 wherein said selectively
providing of said aerobic microbial consortium comprising of microbes comprises obtaining
the same from municipal sewage through enrichment in nitrate broth and with said
Escherichia coli DHl and Pseudomonas sp BWDY-42 and/or microbes having substantial
identity with said Escherichia coli DHl and Pseudomonas sp BWDY-42 in the proportion of
about 82:18.
9. A process for producing the biofertilizer as claimed in anyone of claims 7 or 8 comprising
step of isolation of said aerobic microbial consortium involving the steps of
a. acquiring municipal sewage followed by addition of a proportion (1%) of the same to the
nitrate broth;
b. repeated cultivation at an optimum temperature preferably at 37 °C; and
c. obtaining therefrom the said aerobic microbial consortium through enrichment.
10. A biofertilizer involving aerobic microbial consortium and its process of manufacture
substantially as herein described and illustrated in reference to the accompanying examples.

A Gyro Mixer machine for mixing of containerized liquid/paint with increased loading
capacity and low power input. Advantageously, the gyro-mixer is adapted for
operation even manually in case of power failures and/or on regions where power
supply is not readily available. The Gyro mixer machine is smaller, and compact in
construction, having an external Rear Pulley facilitating manual operation in case of
power failure. Moreover, the drive system is mounted on a steel construction Main-
Frame ensuring longer operating life free of repair/replacement.