Lecture 2 microbial_sem_6_20180220

Biofer'lizers
Microbial Biotechnology|Biotech-552|Dr. Zia|Lec 2

1.  What are Biofer<lizers?
2.  Advantages of Biofer<lizers
3.  Types of Biofer<lizer
4.  Phosphate solubilizing bacteria
5.  Mechanism of phosphate solubiliza<on
6.  Gene<cs of phosphate solubiliza<on
7.  Bio-inoculants of PSM
8.  Prac<cal screening approach for PSM
9.  Mass produc<on of Biofer<lisers
10. New Trends in Formula<ons Using Unconven<onal
Synthe<c Materials
11. Encapsulated formula<ons
12. Regula<on and control of contamina<on of commercial
inoculants
13. Cost of development and marke<ng
14. Conclusions and future prospects

1.  What are Biofer<lizers?
2.  Advantages of Biofer<lizers
3.  Types of Biofer<lizer
4.  Phosphate solubilizing bacteria
5.  Mechanism of phosphate solubiliza<on
6.  Gene<cs of phosphate solubiliza<on
7.  Bio-inoculants of PSM
8.  Prac<cal screening approach for PSM
9.  Mass produc'on of Biofer'lisers
10. New Trends in Formula'ons Using Unconven'onal
Synthe'c Materials
11. Encapsulated formula'ons
12. Regula'on and control of contamina'on of commercial
inoculants
13. Cost of development and marke'ng
14. Conclusions and future prospects

In the 19th century, chemical fer'lizers were
introduced in agriculture. But slowly chemical
fer'lizers started displaying their ill-eﬀects such
as:

•  Leaching out
•  Pollu'ng water basins
•  Destroying micro-organisms and friendly insects
•  Making the crop more suscep'ble to the aRack of diseases
•  Reducing the soil fer'lity and thus causing irreparable
damage to the overall system

What are Biofer'lizers?
Those substances that contain live microbes
which helps in enhancing the soil fer<lity either
by ﬁxing atmospheric nitrogen, solubiliza<on of
phosphorus or decomposing organic wastes or
by augmen<ng plant growth by producing
growth hormones with their biological ac<vi<es.

Advantages of Biofer'lizers
•  Renewable source of nutrients
•  Decompose plant residues, and stabilize C:N ra'o of soil
•  Improve texture, structure and water holding capacity of soil
•  S'mulates plant growth by secre'ng growth hormones
•  Secrete fungi sta'c and an'bio'c like substances
•  Solubilize and mobilize nutrients
•  Eco-friendly, non-pollutants and cost eﬀec've method
•  Increase crop yield by 20-30%
•  Replace chemical nitrogen and phosphorus by 25%
•  Provide protec'on against drought and some soil borne
diseases

Types of Biofer'lizer

Types of Biofer'lizer
For nitrogen
•  Blue –Green Algae (BGA) and Azolla for low land paddy
For phosphorus
•  Celluloly<c fungal culture

Phosphate solubilizing bacteria
§  17 essen'al plant nutrients (“Liebig’s law of the minimum”, Emanuel Epstein,2004)
§  Phosphorous is second only to nitrogen in nutrients for plants (Glass et al.,1989 and
vance 2001)
§  Phosphorus make up about 0.2 % for plant dry weight (Whitelaw et al.,1999)
Limita'ons
§  Lack of large biological source (Ezawa et al. 2002).
§  Immobiliza'on occurs through precipita'on reac'on with highly reac've Al3+ ,Fe3+
and Ca2﹢(Gyaneshwar et al., 2002; Hao et al., 2002).
§  The monobasic (H2PO4
−) and the diabasic (HPO4
−2) ions, only soluble forms (Glass, 1989).
§  Eﬃciency of P fer'lizer throughout the world is around 10 - 25 % (Isherword, 1998).
§  0.1% of the total P exists in a soluble form available for plant uptake (Zou et al. 1992).

§  Rapid precipita'on of P, in the form of Fe–P and Al–P complexes (Gyaneshwar et al. 2002,
Rengeland Marschner 2005 ,Johnson and Loepper 2006).

§  P fer'lizers produc'on - energy intensive treatment with sulfuric acid at high
temperature (Vassilev et al. 2006).
§  Use of PSMs can increase crop yields up to 70 percent (Verma, 1993). Megatherium
var.Phospha8cum was ﬁrst used in Russia in 1953 in a fer'lizer called Phosphobacterin (Cooper,
1959).
§  Applica'on of several 'mes more phosphorus than the plant needs (Nyle and
Ray RW ,1999).
§  Microorganisms enhance the P availability to plants by mineralizing organic and inorganic P in soil
and by solubilizing precipitated phosphates (Chen et al., 2006; Kang et al., 2002; Pradhan and
Sukla, 2005).
Drawbacks of chemical fer'lizers (p)
Phosphate solubilizing microorganisms

§  Strains from the genera Pseudomonas, Bacillus and Enterobacter are among the most
powerful phosphate solubilizers (Rodriguez et al. 1999, Whitelaw,2000, Igual et al., 2001,Ahmad Ali Khan et al., 2010)
§  The PSB strains exhibit in organic P-solubilizing abilities ranging between 25–42 µg P
mL−1 and organic P mineralizing abilities between 8–18 μg P mL−1 (Tao et al., 2008).
Phosphate solubilizing Bacteria (PSB)
§  Bacteria are more effective in phosphorus solubilization than fungi (Alam et al., 2002)
§  Whole microbial population in soil, PSB constitute 1 to 50 %, while phosphorus
solubilizing fungi (PSF) are only 0.1 to 0.5 % in P solubilization potential (Chen et al., 2006)
Phopshate solubilizing Fungi (PSF)
• Aspergillus, Penicillium (Antoun et al. 1998; Buch et al. 2008; Gulati et al. 2008; Son et al. 2006;
Sulbarán et al. 2009; Whitelaw 2000). Arbuscular mycorrhizae and Piriformis indica (Waller et al., 2005).

Mechanism of phosphate solubiliza'on
chelation-mediated mechanism
§ Low molecular weight metabolites release by PSM such as organic acids.
§ Decrease in the pH of basic soils.
§ Hydroxyl and carboxyl groups of acids chelate cations (Al, Fe, Ca) bound to
phosphate and then converted into soluble forms (He and Zhu, 1988 ; Kpomblekou and
Tabatabai 1994; Goldstein, 1995 ; Surange, 1995; Dutton and Evans, 1996; Nahas, 1996; Vázquez P. México.1996; Rudolfs. 1996 ;
Sagoe et al., 1998 ; Deubel et al., 2000 ; Whitelaw, 2000 ; He et al., 2002 ; Stevenson, 2005 ; Fankem et al., 2006).
§ Gluconic acid 2-alpha-ketogluconic acid seems to be the most frequent agent of mineral
phosphate Solubilization (Illmer et al,.1992,Gupta et al,.1994,Liu et al,. 1992,Goldstein et al,.1995) .
Solubilization of mineral phosphates
Solubilization of organic phosphate
§  Phosphatases (also called phosphohydrolases).
§  Cleavage of C-P bonds from organophosphonates (Ohtake, H.
1996; McGrath, J.W. 1995; McGrath, J.W. 1998; Bujacz, B. 1995).

Gene'cs of phosphate solubiliza'on
•  Phosphate-repressible phosphatases e.g. sudden and full induction of phoA
gene of E. coli by decreasing Pi concentration from 100 mM to 0.16 mM.
•  Phosphate-irrepressible production e.g. Morganella morganii and Providencia
stuartii .
•  Temperature-dependent phosphatase enzymes e.g. Pseudomonas
fluorescens expression of apo gene, which encodes for anacidic phosphatase
enzyme at transcriptional level was maximal at 17.5°C.
•  Type of carbon source regulated acid phosphatases e.g. p27 enzyme (acid
phosphatase, class B) in E. coli MG1655 was switched off when cells were
grown on glucose while it was turned on when a source of carbon other than
glucose was used.
Genetics of organic phosphate mineralization

Bio-inoculants of PSM
Carriers/Medium
• Bentonite, corn oil, mineral soils, peat, peat moss, rice
straw,vermiculite, and perlite(volcanic stone composed of little
hydrated aluminium silicate)
• Sterilized mill peat with natural graphite, calcium carbonate,
and water based medium containing sucrose and yeast
extract (Novozymes BioAg Group formulation).
Beneficial microbes + Carriers/Medium
Single culture approach (SCA)
Multiple or mixed culture approach (MCA)

Prac'cal screening approach for PSM
Purification 4x
Confirmation of activity on NBRIP
Gram staining
whole cell PCR-16SrDNA sequencing
Gel electrophoresis
Sending for sequencing
Identification using gene bank
Biochemical analysis
Glucose, 10 g; Ca3 (PO4)2, 5 g; MgCl2.6H2O, 5 g;
MgSO4.7H2O, 0.25 g; KCl, 0.2 g, and (NH4)2SO4,
0.1 g

Purification 4x
Alcian blue staining
PCR amplification of ITS 1 and ITS 2 region
Gel electrophoresis
Blast analysis
Purification 4x
Gram staining
whole cell PCR-16SrDNA sequencing
Gel electrophoresis
Identification using gene bank
Biochemical analysis
Glucose, 10 g; Ca3 (PO4)2, 5 g; MgCl2.6H2O, 5 g;
MgSO4.7H2O, 0.25 g; KCl, 0.2 g, and (NH4)2SO4,
0.1 g

Solubilization index
PSI 2.5 2.3 2.6


Bacillus subtilis Escherichia coli
Strain CStrain A Strain B
Gram staining

Tests Ac've
Ingredients

Reac'on/Enzymes Colors Results

ONPG 2-nitrophenyl-
ßDgalactopyranoside
ß-galactosidase Colorless -
ADH L-arginine Arginine Dihydrolase Orange +
LDC L-lysine Lysine Decarboxylase Yellow -
ODC L-ornithine Ornithine Decarboxylase Yellow -
CIT trisodium citrate Citrate u<liza<on Blue +
H2S sodium thiosulfate H2S produc<on Greyish -
URE urea Urease Yellow -
TDA L-tryptophane Tryptophan Deaminase Reddish brown +
IND L-tryptophane Indole produc<on Yellow -
VP sodium pyruvate Voges Proskauer reac<on Colorless -
GEL gela<n Gelan<nase Black pigment +
GLU D-glucose Fermenta<on/oxida<on Yellow +
MAN D-mannitol Fermenta<on/oxida<on Blue green -
INO inositol Fermenta<on/oxida<on Blue green -
SOR D-sorbitol Fermenta<on/oxida<on Blue green -
RHA L-rhamnose Fermenta<on/oxida<on Blue green -
SAC D-sucrose Fermenta<on/oxida<on Blue green -
MEL D-melibiose Fermenta<on/oxida<on Blue green -
AMY amygdalin Fermenta<on/oxida<on Blue green -
ARA L-arabinose Fermenta<on/oxida<on Blue green -
Nitrate
reduc<on
(GLU)
Potassium nitrate NO2 produc<on ,reduc<on
to N2 gas
Red +
Biochemical tests
API 20E identification kits (bioMérieux Deutschland GmbH)
Profile id = 2226006
89.6% similarity with
Pseudomonas aeruginosa

Purified DNA for sequencing to
sequencing companies

16S ribosomal databases
•  EzTaxon-e
•  Ribosomal Database Project
•  SILVA
•  GreenGenes

Fungus
Transparent area due to Phosphate solubilization around an
unknown fungus colony
Alcian blue staining of fungus featuring round shape conidia and
phialides
Phosphate solubilizing index = 2.8

unknown
Penicillium chrysogenum
PM1Contig291

The decline in pH observed during phosphate solubilization is inversely
proportional with the concentration of released P (Chen et al. 2006; Hwangbo et al. 2003; Kim et
al. 1997),
Effect of pH on phosphate solubilization

Fermentation Profile of Penicillium chrysogenum

Mass produc'on of Biofer'lisers
•  The culture is incubated un<l maximum cell
popula<on of 1010 to 1011 cfu/ml is produced.
o  Under op<mum condi<ons this popula<on level could be agained with
in 4 to 5 days for Rhizobium; 5 to 7 days for Azospirillum; 2 to 3 days for
phosphobacteria and 6-7 days for Azotobacter.
•  The culture obtained in the ﬂask is called starter
culture. For large scale produc<on of inoculant,
inoculum from starter culture is transferred to large
ﬂasks/seed tank fermentor and grown un<l required
level of cell count is reached.

Prepara'on of carrier material
•  The carrier material (peat or lignite (oken referred to as brown
coal, is a sok brown combus<ble sedimentary rock formed from naturally compressed peat. It is considered the lowest rank
of coal due to its rela<vely low heat content) is powdered to a ﬁne
powder so as to pass through 212 micron
sieve.
•  The pH of the carrier material is neutralized
with the help of calcium carbonate (1:10
ra<o) , since the peat soil / lignite are acidic in
nature ( pH of 4 - 5).
•  The neutralized carrier material is sterilized in
an autoclave to eliminate the contaminants.

Prepara'on of Inoculants
•  The neutralized, sterilized carrier material is
spread in a clean, dry, sterile metallic or
plas<c tray.
•  The bacterial culture drawn from the
fermentor is added to the sterilized carrier
and mixed well by manual (by wearing sterile
gloves) or by mechanical mixer.
•  The culture suspension is to be added to a level of 20 – 30% water holding
capacity depending upon the popula<on.

New Trends in Formula'ons Using Unconven'onal Synthe'c Materials
•  The concept underlying immobilized microbial cells, is to entrap beneficial
microorganisms into a matrix.

Polymers have demonstrated poten<al as bacterial carriers that offered
substan<al advantages :

§  These formula<ons encapsulate the living cells protect the microorganisms against
many environmental stresses, and release them to the soil, gradually but in large
quan<<es.
§  when the polymers are degraded by soil microorganisms, usually at the <me of seed
germina<on and seedling emergence.
§  They can be stored dried at ambient temperatures for prolonged periods, offer a
consistent batch quality and a beger defined environment for the bacteria, and can
be manipulated easily according to the needs of specific bacteria.
§  These inoculants can be amended with nutrients to improve the short-term survival
of the bacteria upon inocula<on

Encapsulated Formula'ons

•  The encapsula<on of microorganisms into a polymer matrix is
s<ll experimental in the ﬁeld o f bacterial-inocula<on
technology.
•  At present there is no commercial bacterial product using this
technology.

•  The formula<on (bacteria-matrix) is then fermented in a
bacterial growth medium.
•  These formula<ons can produce many useful compounds for
industrial and environmental applica<ons (such as organic
acids, amino acids, enzymes) and biodegrade toxic materials
(bio remeda<on) over extended periods of <me.

BoRom LINE

The main goal of these industrial formula<ons is to maintain the cells entrapped
in an ac<ve form for as long as possible. Any premature release of the
microorganisms from these encapsulated forms is undesirable.

REGULATION AND CONTROL OF CONTAMINATION OF COMMERCIAL INOCULANTS
The required level of bacteria cannot be established as a general standard because it
varies from one bacterial species to another. Only rhizobial inoculants have legally
established standards.

Quality control methods to determine the number of bacteria
within the inoculant are not standardized either. To measure the bacterial
number, commonly known methods in microbiology are used; the tradi<onal
Plate Count methods, Most Probable Number.

Examples of New Commercial Microbial Inoculants
Commercial microbial inoculants of other beneﬁcial microorganisms have
begun to appear on the market on a small scale. These include "Azogreen",
a French-approved Azospirillum inoculant.

COST OF DEVELOPMENT AND MARKETING
The cost of developing a new product by the agrochemical industry has been
es<mated at over $80 million US and rising . The development of resistance to
pes<cides may shorten the commercial life of these products and thus their
poten<al return. The development of bacterial inoculants is claimed to be
cheaper than that of agrochemicals.

The following are some factors that reduce the costs of development of
bacterial inoculants which makes them aRrac've to the agrochemical
industry:

(i) Reduced registra<on costs compared to those of
chemical-product test programs that are well- established and costly.

(ii) Reduced registra<on <me decreases the <me span from ﬁrst screening to
market, thus increasing revenues

(iii) The possibility of developing bacterial products for small markets. Since the
cost involved in bringing a new chemical to the marketplace is so large, the
product must be targeted to a market large enough to have a good return
on investment. This limits the choice of crops to the major crops only.

Other mo<va<onal steps for the agrochemical industry to develop
bacterial inoculants might be:
•  It is less likely that pathogens will develop resistance as fast as they do to
chemical products.
•  Some bacterial inoculants, especially those that use an organism
employing a single mechanism against the pathogen, can also develop
resistance.

•  They are "environment friendly". The "natural“ tag of bacterial inoculants
(especially those that are non engineered and indigenous) make them
more acceptable in the public eye, and especially t o the "Green
movement" pressure groups, than chemicals.

Market requirement

First, all the considera<ons men<oned above (efficient strains, op<mized
formula<ons, cost-effec<ve produc<on, and good and prac<cal inocula<on
techniques) are not sufficient to launch a new product on the market nor guarantee
its success. The following prac<cal variables should be considered:

(i) The product must be efficient and reliable in large-scale field trials and especially
under "real life" condi<ons.

(iii) Obviously, patents on industrial processes and registra<on of biological products
must be secured.

(iv) For every poten<al customer country, a market survey must be done which
examines customer demand, market size, and expected selling price.


CONCLUSIONS AND FUTURE PROSPECTS
ü  The agrochemical industry is more sympathe<c now to the concept of
bacterial inoculants than it has been previously. There is a genuine
interest in developing bacterial products that are reliable and that can act
as complements to chemicals already on the market
ü  Greenhouse crops are also primary targets for commercial inoculants
ü  Pioneering transgenic plants are already in the ﬁeld expressing
insec<cidal proteins of B. thuringiensis in cogon plants, making
them resistant to various insect pests.
ü  A gradual and modest increase in the use of bacterial inoculants is to be
expected.
ü  Agriculture in developed countries is deﬁnitely the major promoter of
microbial inoculants that are "environmentally friendly“.

Lecture 2 microbial_sem_6_20180220

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