R.R.

Group Of Modern Technology

NH-24,Bakshi Ka Talab, Sitapur Road , Lucknow

, Uttar Pradesh 227202

A PROJECT ON
“FORMATION OF BIO-FERTILIZER FROM GENETICALLY MODIFIED
MICROBIAL CONSORTIUM”
 Name : Shiksha Maurya
Roll no : 1836154048
Department of Biotechnology, RRIMT
(2020)

Under the guidance

Of
MRD LIFE SCIENCES

Submitted To : MRD LIFE SCIENCES Pvt. Limited

Lucknow, b-3/226010 46&47,2 nd floor , near state bank of India , Vibhuti khand , Gomati Nagar

Lucknow ,Uttar Pradesh website : www.mrdlifesciences.com

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 ACKNOWLEDGEMENT
It was a great pleasure for me to get an opportunity to work for my training at the
MRD LIFE SCIENCESS. It has given me the research experience that will be
definitely helpful in my future career.
On the successful completion of my training, I would like to express my
gratitude to those without whom this training would not have been materialized.
With an overwhelming sense pride and genuine and personal regards to Mr.
Manoj Kumar Verma for his valuable advice and encouragement.
With an overwhelming sense pride and personal regards to my supervisor Mr.
Manoj Kumar Verma, Director of MRD LIFE SCIENCES, Lucknow for her
constructive criticism, valuable advice, close supervision, healthy
encouragement, utmost patience and generosity throughout the course of this
study. Furthermore I would like to thank Mr. Chitranshu Pandey.
Additionally, I would like to thank, Mr. Raj Shekhar Mishra, Ms. Pallavi
Sharma ,MRD Life Sciences Lucknow.
I would like to thank my parents who have supported me throughout entire
process, all by Keeping me harmonious and helping me putting pieces together. I
will be grateful forever for your love and support.

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 CONTENTS

S.No. Title

Introduction
1.

Review of Literature
2.

Objectives
4.

5. Methodology

6.
Result

7. Discussion

8. Conclusion

9. References

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 Abstract
Name : Shiksha Maurya
Branch : Biotechnology (2nd year)
College : R.R I M T Sitapur Road Lucknow up. India
Date :
Page : 50

Plants nutrients are essential for the production of crops and healthy food for the world’s
ever population. Soil management strategies today are mainly dep increasing endent on
inorganic chemical-based fertilizers, which cause a serious threat to human health and
the environment. Bio-fertilizer has been identified as an alternative for increasing soil
fertility and crop production in sustainable farming. The exploitation of beneficial
microbes as bio-fertilizers has become of paramount importance in agricultural sector
due to their potential role in food safety and sustainable crop production.

Bio-fertilizer can be an important component of integrated nutrients management.

Microorganisms that are commonly used as bio-fertilizer components include; nitrogen
fixers (N-fixer), potassium and phosphorus solubilizers, growth promoting rhizobacteria
(PGPRs), endo and ecto mycorrhizal fungi, cyanobacteria and other useful microscopic
organisms. The use of bio-fertilizers leads to improved nutrients and water uptake, plant
growth and plant tolerance to abiotic and biotic factors. These potential biological
fertilizers would play a key role in productivity and sustainability of soil and also in
protecting the environment as eco-friendly and cost effective inputs for the farmers.

Bio-fertilizer contains microorganisms which advance the sufficient supply of

supplements to the host plants and guarantee their legitimate improvement of
development and regulation in their physiology. Bio-Fertilizers are eco-accommodating,
one of the best current tool for agriculture and are utilized to enhance the fertility,
quality and nature of the soil.
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 INTRODUCTION
“Despite the many accomplishments of mankind, we owe our existence to six-inch of top soil and fact
that it rains.” – Confucius

A biofertilizer (also bio-fertilizer) is a substance which contains living micro-

organisms which, when applied to seeds, plant surfaces, or soil, colonize the
rhizosphere or the interior of the plant and promotes growth by increasing the
supply or availability of primary nutrients to the host plant.[1] Biofertilizers add
nutrients through the natural processes of nitrogen fixation,
solubilizing phosphorus, and stimulating plant growth through the synthesis
of growth-promoting substances. The microorganisms in biofertilizers restore
the soil's natural nutrient cycle and build soil organic matter. Through the use
of biofertilizers, healthy plants can be grown, while enhancing the
sustainability and the health of the soil. Biofertilizers can be expected to
reduce the use of synthetic fertilizers and pesticides, but they are not yet able
to replace their use. Since they play several roles, a preferred scientific term
for Conventional agriculture plays a significant role in meeting the food
demands such beneficial bacteria is "plant-growth promoting rhizobacteria"
(PGPR).

BIOFERTILIZER TODAY
Biofertilizers provide "eco-friendly" organic agro-input. Biofertilizers such
as Rhizobium, Azotobacter , Azospirilium and blue green algae (BGA) have
been in use a long time. Rhizobium inoculant is used for leguminous
crops. Azotobacter can be used with crops
like wheat, maize, mustard, cotton, potato and other vegetable rich in various
crops. Azospirillum inoculations are recommended mainly
for sorghum, millets, maize, sugarcane and wheat. Blue green
algae belonging to a
general cyanobacteria genus, Nostoc or Anabaena or Tolypothrix or Aulosira,
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fix atmospheric nitrogen and are used as inoculations for paddy crop grown
both under upland and low-land conditions. Anabaena in association with
water fern Azolla contributes nitrogen up to 60 kg/ha/season and also
enriches soils with organic matter. seaweeds are types of mineral elements
(potassium, phosphorus, trace elements etc) hence they are extensively used
as manure by people of coastal districts. Seaweed - manure also helps in
breaking down clays. Fucus is used by Irish people as manure on a large
scale. In tropical countries bottom mud of dried up ponds which contain
abundant blue green algae is regularly used as manure in fields. The mixture
of seaweeds and blue green algae may serve as ideal fertilizer.

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 History of bio-fertilizer

Bio-fertilizers such as Rhizobium, Azotobacter, Azospirillum and Blue green

algae (BGA) have been in use a long time ago. The knowledge of applied
microbial inoculum is a long history which passes from generation to
generation of farmers. It started with culture of small scale compost
production that has evidently proved the ability of bio-fertilizer [3]. This is
recognized when the culture accelerates the decomposition of organic
residues and agricultural by-products through various processes and gives
healthy harvest of crops [18]. The commercial history of bio-fertilizer began
with the launch of “Nitragin” by Nobbe and Hilther in 1895. This was followed
by the discovery of Azotobacter and then Blue-green algae and a host of other
microorganisms which are being used till date as bio-fertilizer [19].

In Malaysia, industrial scale production of microbial inoculants started in late

1940s and picking up in 1970s taking guide by Bradyrhizobium inoculation
on legumes. Government Research Institute, the Malaysian Rubber Board
(MRB) had been conducting research on Rhizobium inoculums for
leguminous crops in the inter rows of young rubber trees in large plantation.
Besides, University Putra Malaysia (UPM) also conducted many researches
since 1980s on Mycorrhiza and initiated the research to evaluate the
contribution of nitrogen from Azospirillum to oil palm seedling [18]. Bio-
fertilizers are usually prepared as carrier based inoculants containing
effective microorganisms [9]. Microorganisms used as bio-fertilizer include:
Nitrogen fixers (N. fixers) e.g., Rhizobium Spp., Cyanobacteria, and
Azotobacter chroococcum, potassium solubilizers (K – solubilizers) e.g.,
Bacillus mucilaginous, phosphorus solubilizers (P – solubilizers) e.g., Bacillus
megaterium, Aspergillus fumigatus, Plant Growth Promoting Rhizobacteria
(PGPR), Vesicular Arbuscular Mycorrhiza (VAM) e.g., Glomus mosseae and
sulfur oxidizers (S – oxidizers).

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 Advantages and Disadvantages of Biofertilizers
A biofertilizer is not just any organic fertilizer or manure. It consists of a carrier medium
rich in live microorganisms. When applied to seed, soil or living plants, it increases soil
nutrients or makes them biologically available. Biofertilizers contain different types of
fungi, root bacteria or other microorganisms. They form a mutually beneficial or
symbiotic relationship with host plants as they grow in the soil. Biofertilizers have many
advantages and a few disadvantages.

Sustainability

Biofertilizers increase the nitrogen and phosphorus available to plants more naturally
than other fertilizers.The different varieties available allow growers to tailor the
microorganisms used to the needs of particular plants. Biofertilizers are simple to use,
even for novice small growers. Biofertilizers do not pollute the soil or the environment,
whereas chemical fertilizers often result in too much phosphate and nitrogen in the soil.
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The excess then leaches into lakes and streams through runoff. Waters decline in
quality and suffer from overgrowth of algae and the death of fish.

Affordability

Biofertilizers reduce dependence upon expensive petroleum sources of chemical

fertilizers. According to the "Journal of Phytology," demand for chemical fertilizers will
exceed the supply by more than 7 million tons by 2020. The shortage of fossil fuels to
produce chemical fertilizers may drive up prices beyond the reach of small users.
Biofertilizers are a cheap, easy-to-use alternative to manufactured petrochemical
products.

Improved Soil

Biofertilizers restore normal fertility to the soil and make it biologically alive. They boost
the amount of organic matter and improve soil texture and structure. The enhanced soil
holds water better than before. Biofertilizers add valuable nutrients to the soil,
especially nitrogen, proteins and vitamins. They take nitrogen from the atmosphere and
phosphates from the soil and turn them into forms that plants can use. Some species
also produce natural pesticides.

Improved Plants

Biofertilizers increase yield by up to 30 percent because of the nitrogen and

phosphorus they add to the soil. The improvement in soil texture and quality helps
plants grow better during periods of drought. Biofertilizers help plants develop stronger
root systems and grow better. Biofertilizers also reduce the effects of harmful
organisms in the soil, such as fungi and nematodes. Plants resist stress better and live
longer.

Disadvantages

Biofertilizers require special care for long-term storage because they are alive. They
must be used before their expiration date. If other microorganisms contaminate the
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carrier medium or if growers use the wrong strain, they are not as effective. The soil
must contain adequate nutrients for biofertilizer organisms to thrive and work.
Biofertilizers complement other fertilizers, but they cannot totally replace them.
Biofertilizers lose their effectiveness if the soil is too hot or dry. Excessively acidic or
alkaline soils also hamper successful growth of the beneficial microorganisms;
moreover, they are less effective if the soil contains an excess of their natural
microbiological enemies. Shortages of particular strains of microorganisms or of the
best growing medium reduce the availability of some biofertilizers.

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 Review of Literature
Biofertilizers refer to preparations containing primarily active strains of

microorganisms in sufficient number and are capable of fixing either atmospheric

nitrogen or solubilizing phosphorus which is otherwise not available to growing

plants. The literature pertaining to the research problem entitled, “Integrated

nutrient management in cauliflower (Brassica oleracea var. botrytis) and cabbage

((Brassica oleracea var. capitata)” has been reviewed under the following subheads:

2.1 Effect of Azospirillum inoculation on the growth, yield and quality of vegetables.

2.2 Effect of Azotobacter inoculation on the growth, yield and quality of vegetables.

2.3 Effect of VAM (Vesicular Arbuscular Mycorrhizae) inoculation on the growth, yield
and quality of vegetables.

2.4 Effect of PSB (Phosphorus Solublizing Bacteria) inoculation on the

growth, yield and quality of vegetables.

How biofertilizers work?

> Biofertilizers fix atmospheric nitrogen in the soil and root nodules of

legume crops and make it available to the plants.

>They solublize the insoluble forms of phosphates like tricalcium, iron, and

aluminium phosphates into available forms.

> They scavenge phosphate from soil layers.

> They produce hormones and antimetabolites which promote root growth.

> They decompose organic matter and help in mineralization in soil.

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 Effect of Azospirillum inoculation on the growth, yield and quality of
vegetables

Amirthalingam (1988) studied the effect of Azospirillum, nitrogen and NAA on growth
and yield of chilli (Capsicum annuum)

and noticed that inoculation of Azospirillum to seed, soil and seedling increased the
plant height, number of branches, length of tap root and root spread, induced earliness
in the first flower appearance and 50 per cent flowering.

The same treatment increased the number of flowers, number of fruits per plant, fresh
and dry weight of fruit/plant, length and girth of fruit, number of seeds and weight of
seeds/fruit. The dry matter production was increased due to the inoculation of
Azospirillum to seed, soil and seedling.

The same treatment also recorded the highest ascorbic acid content and capsaicin
content. Inoculation of Azospirillum also enhanced the nitrogen and phosphorus content
of the plant.

The yield obtained with the inoculation of Azospirillum to seed, soil and seedling at 75
per cent fertilizer N was on par with the yield obtained by 100 per cent N without
inoculation.

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 TYPE OF BIO-FERTILIZER

Biofertilizers include the following types:

1. Symbiotic Nitrogen Fixers Rhizobium spp.
2. Asymbiotic Free Nitrogen Fixers (Azotobacter)
3. Azospirillum
4. Algae Biofertilizers (Blue Green Algae or BGA in association with Azolla).
5. Phosphate Solubilising Bacteria.
6. Mycorrhizae.

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 Types of Bio-Fertilizers and How they Work

Bio-fertilizers are classified into different types depending on the type or group of
microorganisms they contain. Table 2 shows the classification of bio-fertilizers on the
bases of the different types of microorganisms used. The different types of bio-fertilizers
include:

Table 2: Different Microorganisms used in Bio-fertilizer Production

Groups Examples

Nitrogen fixing bio-fertilizers

Free-living Azotobacter, Bejerinkia, Clostridium,

Symbiotic Rhizobium, Frankia, Anabaena,

Associative symbiotic Azospirillum

Phosphate solubilizing bio-fertilizer

Bacteria Bacillus megaterium var, Phosphaticum, Bacillus subtilis, Bacilluscirculans

Fungi Penicillum Spp. Aspergillus awamori

Phosphate mobilizing bio-fertilizers

Arbuscular Mycorrhiza Glomus Spp., Gigaspora Spp., Acaulospora Spp.

Scutellospora Spp. and Sclerocystis Spp.

Ectomycorrhiza Laccaria Spp. Pisolithus Spp, Boletus Spp. and Amanita

Spp.

Ericoid Mycorrhiza Pezizella ericae

Orchid Mycorrhiza Rhizoctonia solani

Bio-fertilizers for micronutrients

Bacillus Spp Silicate and zinc solubilizers

Plant growth promoting Rhizobacteria

Pseudomonas Pseudomonas fluorescens

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➢ Nitrogen fixing bio-fertilizers (NFB): Examples include Rhizobium Spp., Azospirillum
Spp. and blue-green algae; these work by fixing atmospheric nitrogen and converting
them to organic (plant usable) forms in the soil and root nodules of legumes, thereby
making them available to plants. Nitrogen fixing bio-fertilizers are crop specific bio-
fertlilizers [32].

➢ Phosphate solubilizing bio-fertilizer (PSB): Examples include Bacillus Spp.,

Pseudomoona Spp. and Aspergillus Spp. These work by solubilizing the insoluble forms
of phosphate in the soil, so that plants can use them. Phosphorus in the soil occurs
mostly as insoluble phosphate which cannot be absorbed by plants [33]. However,
several soil bacteria and fungi possess the ability to convert these insoluble phosphates
to their soluble forms. These organisms accomplish this by secreting organic acids which
lower the pH of the soil and cause the dissolution of bound forms of phosphate making
them available to plants [33].

➢ Phosphate mobilizing bio-fertilizers (PMB): Examples are Mycorrhiza. They work by

scavenging phosphates from soil layers and mobilizing the insoluble phosphorus in the
soil to which they are applied. Chang and Yang [34] stated that phosphorus solubilizing
biofertilizer (PSB) sometimes act as phosphate mobilizers. Phosphate mobilizing bio-
fertilizers are broad spectrum bio-fertilizers. Soil phosphorus mobilization and
immobilization by bacteria are shown in Figure 1.

microbiology-biotechnology-reports-phosphorus-mobilization-immobilization

➢ Plant growth promoting bio-fertilizer (PGPB): Examples of plant growth rhizobacteria

are Pseudomonas Spp. etc: these work by producing hormones and anti-metabolites
which promotes root growth, decomposition of organic matter which help in
mineralization of the soil thereby increasing availability of nutrients and improving crop
yield [3,35]. PGPB are crop specific bio-fertilizers.

➢ Potassium solubilizing bio-fertilizer (KSB): Examples include Bacillus Spp. and

Aspergillus niger. Potassium in the soil occurs mostly as silicate minerals which are
inaccessible to plants. These minerals are made available only when they are slowly
weathered or solubilized. Potassium solubilizing microorganisms solubilize silicates by
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producing organic acids which cause the decomposition of silicates and helps in the
removal of metal ions thereby making them available to plants. Potassium solubilizing
bio-fertilizers are broad spectrum bio-fertilizers.

➢ Potassium mobilizing bio-fertilizer (KMB): Example of potassium mobilizing bio-

fertilizer is Bacillus Spp. These work by mobilizing the inaccessible forms of potassium
(silicates) in the soil. Some phosphate solubilizing bio-fertilizers such as Bacillus Spp.
and Aspergillus Spp. has been found to mobilize potassium and also solubilize
phosphorus.

➢ Sulfur oxidizing bio-fertilizer (SOB): Example of sulfur oxidizing microorganism is

Thiobacillus Spp. These work by oxidizing sulfur to sulfates which are usable by plants.

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 CONTRIBUTION OF BIOFERTILIZERS ON CROP YIELD AND ECONOMICS

Inoculation of AM fungi and AM fungi + Azotobacter led to increase in peduncle length,

flag leaf area, number of grains spike–1 , 250 grain weight, grain and biological yields
plant–1 in wheat. AMF and Azotobacter complement each other and resulted in
improved plant growth [5]. Kader et al. [16] reported that there was 18% increase in
grain yield in wheat due to Azotobacter inoculants over the control. Suri and Choudhary
[10] reported that inoculation with either of 3 VAM cultures with increasing P levels from
50 to 75% of recommended phosphorus dose resulted in consistent and significant
improvement in grain protein content, grain and straw yield and nutrient uptake in
wheat.

Effect of Biofertilizers on Use of Commercial Fertilizers

Integrated treatments with biofertilizer and nitrogen showed better performance in

terms of shoot length by 31.9% compared to separate treatments in wheat (Saber et al.
[18]. Singh et al. [19] observed that combination of Azotobacter strain (Azo-8) along with
urea (60 kg N ha-1 ), FYM (40 kg N ha-1 ), resulted in more than 23 and 36% increase in
shoot fresh and dry weight, 26 and 38% increase in root fresh and dry weight of wheat
crop over control regularly. Mane et al. [20] reported that the application of 125% RDF
(80:40:40 kg NPK ha-1 ) + Azotobacter + PSB recorded significantly higher plant height,
number of effective tillers per plant of wheat than all other treatments. Khandare [21]
reported that soil application of carrier biofertilizer at 10 kg ha-1 and liquid biofertilizer
at 0.625 and 1.25 L ha-1 in combination with 75% NP gave significantly more plant
height in wheat over 75% NP alone at different intervals. These treatments were at par
with 100% NP alone in plant height.

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 LIMITATION OF BIO-FERTILIZER

> Unavailability of suitable strain due to lack of availability of specific strain.

> Unavailability of suitable carrier: As persuitability, the order is peat, lignite,

charcoal, farmyard manure, soil, rice bran.

>Lack of awareness among farmers: They are unaware of the damages caused on

the ecosystem by continuous application of inorganic fertilizer.

> Inadequate human resources and inexperienced staff.

> bio-fertilizer is their nutrient content when compared to inorganic fertilizers. This

might result to deficiency symptoms in plants grown with the bio-fertilizer.

> Environmental constraints: Soil characteristics like salinity, acidity, drought water
logging affects the use of biofertilizers

The most important limitation of bio-fertilizer is their nutrient content when compared to
inorganic fertilizers. This might result to deficiency symptoms in plants grown with the
bio-fertilizer. However, this problem can be curbed by the addition of substances such as
bone meal (rich in phosphorus), wood ash (rich in potassium) or other substances of
natural origin such as phosphate rock to enrich the fertilizer. Also the use of nutrient rich
wastes such as palm wastes (rich in potassium), wood ash (rich in potassium also) in
making bio-fertilizer can help to remedy the problem. Mahimairaja et al.

[24] stated that the addition of phosphorus to wastes makes the bio-fertilizer more
balanced and reduces nitrogen losses. Again storage of bio-fertilizer goes a long way in
affecting its efficacy. Even though bio-fertilizer has many positive aspects, its use can
sometimes not lead to the expected positive results and this could be because of
exposure to high temperature or hostile conditions before usage. Bio-fertilizer should be
stored at room temperature or in cold storage conditions away from heat or direct
sunlight and polythene bags used in packaging bio-fertilizer should be of low density
grade with a thickness of about 50 –75 microns [42]. Other constraints limiting the use
of biofertilizer technology may be environmental, human resource, unawareness,
unavailability of suitable strains, and unavailability of suitable carrier and so on [20].
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Short shelf life, lack of suitable carrier material, susceptibility to high temperature,
problem in transportation, and storage are biofertilizers bottlenecks that still need to be
solved in order to obtain effective inoculation [43].

➢ Unavailability of suitable strain due to lack of availability of specific strain: This is one
of the major constrains in the production of biofertilizer. Based on the fact that selected
strains have ability to survive both in the broth and the inoculants carrier.

➢ Unavailability of suitable carrier: If suitable carrier material is not available, it is

difficult to maintain the shelf-life of the bio-fertilizer. As per suitability, the order is peat,
lignite, charcoal, farmyard manure, soil, rice bran.

➢ Lack of awareness among farmers: Farmers are not aware of biofertilizers and their
usefulness in increasing crop yields. They are unaware of the damages caused on the
ecosystem by continuous application of inorganic fertilizer.

➢ Inadequate human resources and inexperienced staff: This is another problem. This
is because the unskilled and the inadequate staff farmers are not given proper
instruction about the application.

➢ Environmental constraints: Soil characteristics like salinity, acidity, drought; water

logging affects the use of bio-fertilizers [20].

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 Caution in the use of Bio-Fertilizers
• Never mix bio-fertilizers with nitrogen fertilizers.

• Never apply bio-fertilizers with fungicides.

• Never expose bio-fertilizers to sunlight directly.

• Bio-fertilizers are stored at room temperature, not below O °C and above 35°C

• Do not keep used solution overnight

Importance of Bio-Fertilizers
Bio-fertilizers play an important role in improving fertility of the soil. In addition, their
application to soil improves the structure of the soil and minimizes the sole use of
chemical fertilizers. Under low land conditions, the application of blue green algae (BGA)
plus Azospirillum proved significantly beneficial in improving yield of grain. Bio-fertilizers
inoculation with Azotobacter and Rhizobium and Vesicular Arbuscular Mycorrhiza gave
the highest increase in straw and grain yield of wheat plants with rock phosphate as
phosphate fertilizer [20]. Azolla is inexpensive, economical, eco-friendly, which provides
benefit in terms of carbon and nitrogen enrichment of soil. It was recorded
microorganisms such as Bacillus subtilis, Thiobacillus thioxidans and Saccharomyces
species can fix atmospheric nitrogen symbiotically and about 80–90% nitrogen demand
could be supplied by soya bean through symbiosis [20].

Bio-control, a modern approach of disease management can be a significant role of bio-

fertilizer in agriculture. Trichoderma based bio-fungicides has been found promising to
control root rot of mung bean [20]. Growth, yield and quality parameters of certain
plants significantly increased with bio-fertilizers containing bacterial nitrogen fixers,
phosphate and potassium solubilizing bacteria and microbial strains of some bacteria .
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The importances of biofertilizers are highlighted below: Secretion of plant growth
hormones which help in plant growth, Protection of the plant against attack by
pathogens, Improvement soil fertility, No special care is necessary while using bio-
fertilizer, Reduction in the use of chemical fertilizers, Bio-fertilizers are cost effective
compared to synthetic fertilizer, Promotes growth of plants, Bio-fertilizers restore the
soil’s natural, nutrient cycle and build soil organic matter and Bio-fertilizer provides
protection against drought.

Microorganisms used in Bio-Fertilizer

Organisms that are commonly used as bio-fertilizers components include nitrogen fixers
(N – fixers), potassium solubilizers (K – solubilizer) phosphorus solubilizer (P –
solubilizer), phosphorus mobilizers (P – mobilizers), used solely or in combination with of
fungi. Most of the bacteria used in bio-fertilizers have close relationship with plant roots.
Rhizobacterium has symbiotic interaction with legume roots, and Rhizobacteria inhabit
root surfaces or rhizosphere soil [3]. The phospho-microorganisms mainly bacteria and
fungi make insoluble phosphorus available to the plants [33]. Several soil bacteria and
few species of fungi possess the ability to covert insoluble phosphate in soil into soluble
forms by secreting organic acids. These acids lower the soil pH and bring about the
dissolution of bound forms of phosphate [33]. While Rhizobium, blue-green algae, and
Azolla are crop specific, bio-inoculants such as Azotobacter, Azospirillum, phosphorus
solubilizing bacteria (PSB), and Vesicular Arbuscular Mycorrhiza (VAM) could be regarded
as broad spectrum bio-fertilizers [33]. VAM are fungi that are found associated with
majority of agricultural crops and enhanced accumulation of plant nutrients

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 METHODOLOGY
Organic farming methods
Natural farming methods
combine scientific knowledge and modern technology with
traditional farming practices based on thousands of years of
agriculture. The distinguishing principle is an avoidance of synthetic
inputs, such as manufactured fertilizers and pesticides, and for this
reason, organic methods are easiest to describe by contrasting them
with conventional, agrichemical - based methods.
In general, organic methods rely on naturally occurring biological
processes, which often take place over extended periods of time,
and a holistic approach, while chemical-based farming focuses on
immediate, isolated effects and reductionist strategies. In
conventional systems, technology hybrid seed, synthetic chemicals,
high-volume irrigation, mechanization is used to regulate local
conditions. Beyond the strictly technical aspects, the philosophy,
day-to-day activities and required skill sets are quite different.

Crop diversity is a distinctive characteristic of organic farming.

Conventional farming focuses on mass production of one crop in
one location, a practice called monoculture. This makes apparent
economic sense: the larger the growing area, the lower the per unit
cost of fertilizer, pesticides and specialized machinery for a single
plant species. The science of agroecology has revealed the benefits
of polyculture (multiple crops in the same space), which is often
employed in organic farming. Planting a variety of vegetable crops
supports a wider range of beneficial insects, soil microorganisms,

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and other factors that add up to overall farm health, but managing
the balance requires expertise and close attention.

Farm size –

Farm size in great measure determines the general approach and specific tools and
methods. Today, major food corporations are involved in all aspects of organic
production on a large scale. However, organic farming originated as a small-scale
enterprise, with operations from under one acre to under 100. The mixed vegetable
organic market garden is often associated with fresh, locally-grown produce, farmers'
markets and the like, and this type of farm is often under 10 acres. Farming at this scale
is generally labor-intensive, involving more more manual labor and less mechanization.
The type of crop also determines size: organic grain farms often involve much larger
acreage. Larger organic farms tend to use methods and equipment similar to
conventional farms, centered around the tractor.

Plant nutrition

Soil fertility –

The central farming activity of - fertilization - illustrates the differences. Organic farming
relies heavily on the natural breakdown of organic matter, using techniques like green
manure and composting, to replace nutrients taken from the soil by previous crops. This
biological process, driven by microorganisms, allows the natural production of nutrients
in the soil throughout the growing season, and has been referred to as feeding the soil
to feed the plant. In chemical farming, individual nutrients, like nitrogen, are synthesized
in a more or less pure form that plants can use immediately, and applied on a man-
made schedule. Each nutrient is defined and addressed separately. Problems that may
arise from one action (e.g. too much nitrogen left in the soil) are usually addressed with
additional, corrective products and procedures (e.g. using water to wash excess nitrogen
out of the soil).

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Pest control –

Differing approaches to - pest control - are equally notable. In chemical farming, a

specific insecticide may be applied to quickly kill off a particular insect pest. Chemical
controls can dramatically reduce pest populations for the short term, yet by unavoidably
killing (or starving) natural predator insects and animals, cause an ultimate increase in
the pest population. Repeated use of insecticides and herbicides and other pesticides
also encourages rapid natural selection of resistant insects, plants and other
organisms, necessitating increased use, or requiring new, more powerful controls.

Livestock –

Raising livestock and poultry, for meat, dairy and eggs, is another traditional, farming
activity that complements growing. Organic farms attempt to provide animals with
"natural" living conditions and feed. Ample, free-ranging outdoor access, for grazing and
exercise, is a distinctive feature, and crowding is avoided. Feed is also organically grown,
and drugs, including antibiotics, are not ordinarily used (and are prohibited under
organic regulatory regimes). Animal health and food quality are thus pursued in a holistic
"fresh air, exercise, and good food" approach. In conventional livestock operations,
animal needs are identified, isolated, and handled discretely. Drugs and synthetic food
supplements are key components. Animals are often given preventive treatment of
antibiotics in their daily feed, and supplements are added to increase the nutritional
value of a variety of substances used as feed. Hormones may be used to optimize
certain characteristics (e.g. produce more meat, or more milk). Living conditions are
often set as the minimum necessary for survival and growth.

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Methodology of Bio-fertilizer

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RESULTS
Effect of biofertilizers

Biofertilizers increased plant productivity in both pot and field studies by an average of 18.4% and 15.3%,
respectively (Fig. 1). Inoculation had more variable effects in pot studies, with yield increases ranging from 9.2%
to 28.7% as compared to 12.5% to 18.1% in field studies. Although productivity gains were 3% greater under
controlled conditions than in the field, this difference was not statistically significant.

Figure 1.

Biofertilizers increase maize yield by an average of 15.3% in the field and 18.4% in greenhouse studies using
field soil. Data labels in parentheses represent the lower and upper bounds of the 95% confidence interval, and
axis labels in parentheses indicate the number of studies and number of observations included in that category
after correcting for publication bias. Effect sizes and confidence intervals were generated by bootstrapping with
4999 iterations.

Climate

Climate region according to the Köppen classification system influenced biofertilizer efficacy in the field but not
in greenhouse studies (Fig. 2). Biofertilizers were twice as effective in arid steppe climates (BS) as in equatorial
climates (Aw) and three times as effective in arid steppe climates (BS) as in fully humid snow climates (Df). Mean
effect sizes were consistent between field and pot studies in arid steppe and desert climates (BS and BW), but
lower in the field in equatorial climates with dry winters (Aw). Effect sizes were more variable in pot studies for all
climate regions.

Figure:2

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Climate affects biofertilizer performance only under field conditions, where biofertilizers are more effective in
arid climates than snow climates. Climate classifications and names are according to the Köppen system, with
temperature (T) and precipitation (P) criteria defined for each region as follows: Aw: Tmin ≥ 18°C and Pmin < 60
mm in winter; BS: 5 Pth < Pann < 10 Pth; BW: Pann ≤ 5 Pth; Cf: −3°C ≤ Tmin ≤ +18°C and not satisfying the criteria of
Cs or Cw; Cw: −3°C ≤ Tmin ≤ +18°C and Pwmin < Psmin and Psmax > 10Pwmin; Df: Tmin ≤ −3°C and not satisfying the
criteria of Ds or Dw. Pth is a precipitation threshold defined based on mean annual temperature, Pwmin is
minimum winter precipitation, Psmin is minimum summer precipitation and Psmax is maximum summer
precipitation (Kottek et al. 2006). Data labels in parentheses represent the lower and upper bounds of the 95%
confidence interval, and axis labels in parentheses indicate the number of studies and number of observations
included in that category after correcting for publication bias. Effect sizes and confidence intervals were
generated by bootstrapping with 4999 iterations.

Soil fertility

Starting soil N content influenced the outcome of inoculation under controlled conditions but not in the field
(Fig. 3A). Inoculation in pot studies was more effective and widely variable when starting soil N was low (below
25 ppm inorganic N or 0.1% total N), increasing yields by 153% as compared to 17.1%, although only three
studies were represented in the low-N dataset. Interestingly, fertilizer application within the growing season did
not alter the outcome of inoculation in a consistent manner in field and pot studies (Fig. 3B).

Figure 3.

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Pre-plant soil fertility affects biofertilizer performance only under controlled conditions. (A) Yield gains due to
biofertilizer inoculation are largest when pre-plant soil nitrogen is below 25 ppm inorganic N or 0.1% total N in
pot studies, but unaffected by pre-plant N in the field. (B) Fertilization during the growing season does not
significantly influence biofertilizer efficacy. Data labels in parentheses represent the lower and upper bounds of
the 95% confidence interval, and axis labels in parentheses indicate the number of studies and number of
observations included in that category after correcting for publication bias. Effect sizes and confidence intervals
were generated by bootstrapping with 4999 iterations.

Taxonomy and ecological coherence

Meta-analyses within subcategories of phylum and genus suggest that ecological coherence may exist at both levels.
Proteobacteria were more effective than Firmicutes in the field, while inoculation with strains belonging to the Bacteroidetes
did not consistently improve maize yields (Fig. 4A). Under controlled conditions, yield increases due to Firmicutes and
Proteobacteria were not significantly different.

Figure 4.

Taxonomy influences biofertilizer efficacy only in the field. (A) Strains belonging to the phylum Proteobacteria
were more effective than those belonging to the Firmicutes in the field, and (B) inoculation with Azotobacter
increased maize yields more than inoculation with Bacillus, Burkholderia, Pantoea or Pseudomonas. Data labels
in parentheses represent the lower and upper bounds of the 95% confidence interval, and axis labels in
parentheses indicate the number of studies and number of observations included in that category after
correcting for publication bias. Effect sizes and confidence intervals were generated by bootstrapping with 4999
iterations.

Although relatively few genera included in the meta-analysis were represented by a minimum of three studies,
the greatest taxonomic differences were observed at this level (Fig. 4B). Strains belonging to the
genus Azotobacter increased yields by an average of 34.4% in the field, the largest increase of any genus.
Biofertilizers composed of Azospirillum spp. and Pseudomonas spp. were also consistently effective,
but Burkholderia spp. increased yields by only 5.0% on average. Inoculation with Bacillus spp., Enterobacter spp.
or Pantoea spp. did not always increase yields, as shown by 95% confidence intervals that overlapped 0.

Pseudomonas spp. consistently increased yields in pot studies as well, with a 95% confidence interval of 11.7%–
38.6%. The remaining three genera represented in both types of studies behaved differently under controlled
conditions than in the field. Despite performing consistently well in the field, Azospirillum spp. were not
significantly better than an uninoculated control in pot studies. In contrast, Bacillus spp. and Enterobacter spp.

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were not significantly more beneficial than uninoculated controls under field conditions, but increased yields by
23.0% and 18.6%,respectively, under controlled conditions.

Ecological complexity

Biodiverse consortia of two or more strains were equally as effective as single-strain formulations in both pot and
field studies (Fig. 5). Single inoculant strains (95% CI 11.8%–17.9%) had more consistent effects in the field
than multiple-strain biofertilizers (95% CI 10.4%–24.0%), but both types of biofertilizers were equally variable in
pot studies (single-strain 95% CI 12.1%–34.8%, multiple-strain 95% CI 5.0%–25.4%).

Figure 5.

Multiple-strain biofertilizers tend to outperform single-strain formulations in the field, but not in the greenhouse.
Data labels in parentheses represent the lower and upper bounds of the 95% confidence interval, and axis
labels in parentheses indicate the number of studies and number of observations included in that category after
correcting for publication bias. Effect sizes and confidence intervals were generated by bootstrapping with 4999
iterations.

Plant-growth-promoting mechanisms

Insufficient greenhouse studies were available to directly compare outcomes of field and pot studies. In the field,
only ACC deaminase production affected the outcome of biofertilizer application out of the PGPR mechanisms
investigated here (Fig. 6). Strains that produced ACC deaminase were more effective than those that were not
proven to produce this compound, increasing yields by 18.5% as compared to 11.1%. Inoculation with strains
possessing all other PGPR mechanisms, including P solubilization, N2 fixation, phytohormone synthesis or
siderophore production, was not more beneficial under field conditions than inoculation with strains not proven
to possess these traits.

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 DISCUSSION
Biofertilizer effects on yields and/or biomass tended to be slightly higher and more variable in
greenhouse pot studies using field soil than in the field (Fig. 1). Soil characteristics of the controlled
environment that favor microbial growth and survival, such as higher temperature, more homogeneous
moisture, altered bulk density and decreased investment in root biomass in pots as compared to the
field, may have contributed to the trend towards greater productivity increases in pot studies.
Alternatively, the trend towards higher mean effect size under controlled conditions could be a
consequence of the product development pipeline, as successful in vitro and pot trials typically precede
improvements in field-applied biofertilizers.

Even when restricting the scope of the meta-analysis to greenhouse trials that used unprocessed field
soil rather than sterilized soil or potting media, studies under controlled conditions were relatively poor
predictors of the influence of climate and soil variables on biofertilizer performance. Climate region
significantly affected field study outcomes but not the outcomes of pot studies using field soil, showing
that temperature and precipitation influence the effectiveness of biofertilizers separately from any
climate-driven soil properties (Fig. 2). Our findings that mean effect size was higher in arid climates and
lower in fully humid snow climates are consistent with a recent meta-analysis showing that PGPR may
be more beneficial under water stressed conditions (Rubin, Groenigen and Hungate 2017). Variability
according to climate is also consistent with theoretical understanding of context-dependent resource
mutualisms (Hoeksema and Bruna 2015). It has also been argued elsewhere that plant-growth-
promoting microorganisms provide greater benefit under stressful conditions (Nadeem et al. 2014).
Downregulation of plant stress responses through bacterial production of ACC deaminase, which
modulates ethylene signaling pathways and appears to consistently increase yields (Fig. 6), could
ameliorate yield penalties due to drought and salinity stress in arid climates (Zafar-ul-Hye et al. 2014).
However, the specific mechanisms underlying growth promotion at a given field site likely vary
according to biofertilizer strain and environmental variables.

Soil N levels influenced the effectiveness of biofertilizers under greenhouse conditions but not in the
field. Albeit highly variable, biofertilizers were significantly more beneficial in pot studies when pre-plant
soil N was low (below 25 ppm inorganic N or 0.1% total N), and additional fertilization did not alter their
effectiveness (Fig. 3). No single standard exists for ‘high’ soil N, with some sources recommending no
additional pre-plant fertilization above 19 ppm NO3-N (Schmitt and Randall 1994) and other
fertilization guidelines declining to establish such a threshold (Geisseler 2011). When the analysis was
repeated using a cutoff of 50 ppm, no effect of pre-plant soil N was found (data not shown), suggesting
that there may be a critical threshold of pre-plant soil N above that biofertilizers are less effective.
Unfortunately, given the paucity of data and inconsistency in forms of N reported, a regression to
determine this threshold could not be constructed from the present data set. The relationship between
soil nitrogen, biofertilizers and yield is complex and likely not easily predicted by a single metric.
Discrepancies between greenhouse and field results for soil fertility are unsurprising in light of poor
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greenhouse-to-field correlations reported for plant-soil feedbacks (Heinze et al. 2016) and nitrogen
availability (Michrina, Fox and Piekielek 1981) but highlight the need for a novel approach to
biofertilizer development and testing.

Understanding the extent of ecological coherence in biofertilizers could increase predictive power,
facilitate targeted screening efforts, and help large -omics studies target bacterial taxa known to
improve growth and productivity. At the phylum level, members of the Proteobacteria were more
effective than Firmicutes in the field, but not under controlled conditions (Fig. 4). Proteobacteria are a
large, metabolically diverse phylum of Gram-negative bacteria subdivided into five classes (Marin
2011), of which three were represented in this meta-analysis: Alphaproteobacteria, Betaproteobacteria
and Gammaproteobacteria. Interestingly, the mean effect sizes of each class were identical, although
the Gammaproteobacteria had lower variability (Fig. S1, Supporting Information). Observed patterns of
ecological coherence at the phylum level do not guarantee that all members of a given taxon will
promote plant growth. In fact, the extent of ecological coherence has been shown to vary among taxa
(Koeppel and Wu 2012), which could explain the similar magnitude in variability within a single class of
Proteobacteria and between the Proteobacteria and other phyla. It should also be noted that sampling
bias could generate false positive findings of ecological coherence (Koeppel and Wu 2012), if
researchers study only a few common or easy-to-culture members of any phylogenetic group. Genomic
analysis could be used to confirm whether unstudied, related strains indeed share metabolic
capabilities of interest.

Azotobacter, Pseudomonas and Azospirillum were the most consistently effective genera in the field
(Fig. 4). Both Azotobacter and Pseudomonas are members of the Pseudomonodaceae, a family of
Gammaproteobacteria, while Azospirillum belongs to the Alphaproteobacteria. Specific genes involved
in plant growth promotion are conserved across members of these genera and other Proteobacteria,
including genes controlling ACC deaminase production, N2 fixation and P solubilization (Bruto et al.
2014). The poor performance in the field by Bacillus spp., spore-forming members of the Firmicutes, is
particularly striking given the long history of plant growth promotion and biocontrol studies focusing on
this genus (Pérez-García, Romero and de Vicente 2011; Shafi, Tian and Ji 2017). Bacillus spp.,
Enterobacter spp., and Pseudomonas spp. were all equally effective under controlled conditions.

Unfortunately, lack of data precluded meta-analysis at lower taxonomic levels, so it remains to be seen
whether certain species are globally relevant for maize or whether biofertilizers must be tailored to a
specific climatic and agronomic context. Philippot et al. (2009) observed that it is easier to identify an
effect of ecological coherence when focusing on low taxonomic ranks in the context of spatial
distribution of microbes. However, the underlying rationale may pertain less to plant growth promotion
than to habitat adaptation: shared physiological traits conferring tolerance to salinity, for example, may
be more likely in closely related bacterial taxa (Lozupone and Knight 2007), whereas phylogenetically
distant taxa may have evolved different biosynthetic pathways to produce the same plant-growth-
promoting compound. Phylogenetic meta-analysis taking into account relatedness of the species being
investigated could provide great insight into the question of ecological coherence were sufficient
species-level data available (Lajeunesse 2009).

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Multi-strain biofertilizer formulations represent an intermediate between single-strain inoculation and in
situ enhancement of soil microbial communities, or rhizosphere engineering (Ryan et al. 2009;
Dessaux, Grandclément and Faure 2016). We found that biodiverse consortia of two or more strains
had more variable effects than single-strain formulations in the field, with yield increases of 10.4%–
24.0% (Fig. 5). Single strains may be more predictable than multi-strain formulations, but the
substantial yield increases at the upper end of the observed range suggest untapped potential for
biodiverse consortia tailored to a specific environment. Successful multi-strain consortia would ideally
include strains with complementary mechanisms of plant growth promotion and/or different
preferences in edaphic and climatic conditions to provide multiple benefits under varied conditions.
Emergent biosynthetic capacity is maximized at intermediate functional relatedness (Chiu, Levy and
Borenstein 2014), suggesting that biofertilizers combining microbial strains from different ecological
guilds could synthesize plant-growth-promoting compounds not seen in single-strain formulations.
However, identification of strains that significantly increase plant growth individually and can also be
combined into a shelf-stable product where mutualism and commensalism are the dominant forms of
microbe-microbe interaction poses substantial challenges.

Nonetheless, a shift towards increasing ecological complexity advances biofertilizers beyond a

misplaced conception of microbial products as universally applicable agrochemicals to a more nuanced
understanding of biofertilizer application as tools to manage soil microbial ecology. Soil microorganisms
have evolved over millions of years to become individually adapted to a vast diversity of ecological
niches, and the quest for a single microbial species that will improve plant growth under all conditions
is unlikely to succeed. There is increasing understanding that complex microbiomes are more beneficial
to plants, particularly under abiotic stresses such as salinity, than individual microbial partners (Qin et
al. 2016). Microbial communities are extremely dynamic and adapt rapidly to alterations in
environmental conditions such as moisture to confer adaptive traits to plant hosts (Lau and Lennon
2012). Isolation of indigenous microbial communities adapted to temperature or salinity extremes or
nutrient-poor soils have been shown to improve plant growth in stressful environments (Kaplan et al.
2013), a phenomenon termed habitat-adapted symbiosis (Rodriguez et al. 2008, 2009). Microbial
community-based approaches to inoculum development may thus represent the next advance to
effective biofertilizers.

In vitro evidence of plant-growth-promoting traits does not appear to predict biofertilizer performance in
the field (Fig. 6). Only ACC deaminase synthesis, a trait that has been previously identified as important
in PGPR and described in detail elsewhere (Glick 2014), appears to increase biofertilizer efficacy in the
field. Few studies explicitly measured mechanisms such as P solubilization, phytohormone production,
or N fixation prior to application, but in every case, strains previously identified as possessing these
traits tended to be less effective in the field. Furthermore, while Bacillus strains possessing multiple
plant-growth-promoting traits have been shown to increase root length and weight, shoot biomass and
grain yield of wheat more than single-trait strains (Baig et al. 2012), meta-analysis showed that
multiple traits provided no advantage over single traits in field or pot studies (Fig. S2, Supporting
Information). While traits such as P solubilization and siderophore production are advantageous in
theory (Hayat et al. 2010) and under axenic conditions (Hussain et al. 2013), to the best of our
knowledge, empirical evidence for their success in maize field studies is lacking. Energetically
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expensive symbiont traits such as N2 fixation could represent a net cost to the host plant in a fertile
field, but such a cost-benefit analysis likely does not apply to free-living strains and traits that require
relatively little metabolic investment. This result may instead indicate the need to move from in vitro to
in situ testing of plant-growth-promoting mechanisms, as strains positive for a given trait according to
laboratory tests may fail to express the same trait in the field

CONCLUSION
Biofertilizers present a realistic option for sustainable intensification of maize production
with great potential to increase yields ∼15%–18%. Nonetheless, the results of this meta-
analysis indicate a critical need for pipelines to effectively tap into microbial
communities and better understand factors that affect biofertilizer performance.
Potential biofertilizer strains are frequently tested in laboratory and pot studies to
predict efficacy in the field, but discrepancies with field outcomes for soil, taxonomic,
and mechanistic moderating variables suggest the need to reconsider this approach. As
proposed by Trivedi et al. (2017), biofertilizers should also be integrated with biocontrol
goals, soil amendments and crop traits for different soil types. The complexity of tailoring
this approach to diverse agroecosystems will require a much greater emphasis on
location-specific abiotic and biotic factors than has previously been the case in
biofertilizer development. Reversing the traditional lab-to-field pipeline may therefore
hold better promise for developing effective biofertilizers in an agricultural context.
Sequencing and network analysis of existing soil microbial communities that increase
resilience to abiotic and biotic stressors could identify keystone taxa and interactions
that are conserved across environments. While only the small percentage of
microorganisms that can be cultured could be formulated into biofertilizers, such an
analysis could identify many more candidate strains than are currently known. In the
future, improved understanding of external variables that affect plant-microbe
interactions and an emphasis on microbial ecology over reductionist methods may
facilitate the development of widely relevant, ecologically complex biofertilizers.

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INSTRUMENTS

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