BIO SOIL MAGIC - Environmental XPRT · BIO SOIL MAGIC is intended for use in field crops, horticulture, turf, forage and ornaments. BIO SOIL MAGIC product should be used by persons

BIO SOIL MAGIC ORGANIC SOIL ADDITIVE TO STIMULATE SOIL HEALTH

ASSISTS IN ACHIEVING SUSTAINED PLANT GROWTH AND YIELD

BIO SOIL MAGIC is based on an advanced scientific approach combining the benefits of bio fertilizers and new

technological solutions using naturally occurring biostimulants.

BIO SOIL MAGIC is intended for use in field crops, horticulture, turf, forage and ornaments.

BIO SOIL MAGIC product should be used by persons trained in the proper methods of handling and applying.

Microbes in this product help to maintain the ecological balance and play a pivotal role in improving soil

conditions for agriculture.

CHARACTERISTICS OF BIO SOIL MAGIC:

Can be applied to all crops and edible Fungi

Has no side effects and residue

Natural

Non-chemical

Non-toxic

Safe

SALIENT FEATURES OF BIO SOIL MAGIC:

ASSISTS IN:

Creating non ambient conditions to the pests

Improving the quality of yield.

Making the bud and root strong.

Improving photosynthesis,

Improving the germination percentage

Increasing the output

Making the plant to grow stronger.

Preventing the crop diseases

Promoting plant burgeon earlier

Promoting the seed to sprout

Strengthening the strength of growing

Precautions:

Avoid spraying during bad weather conditions.

If product agglomerated does not affect the results of application.

Avoid chemical pesticides three days before and after applying BIO MAGIC

Recommended Dose & Usage

30 Tabs or 50 gm/Acre in drip or as a drench or using a

sprayer once in 30 days starting from -7 days

(Increasing dosage or frequency properly in disease or

cold district, depleted soil, acidic soil, long growth and

development period, perennial crop)

Dilute 2 gm or 2 tablets with 1L of water for seed soaking

and spray after seeding.

COMPATIBILITY

BIO SOIL MAGIC is incompatible with synthetic

fungicides, chemical insecticides and herbicides.

Avoid spraying during bad weather conditions.

If product agglomerated does not affect the results of

application.

Avoid chemical pesticides three days before and after

applying BIO SOIL MAGIC.

SHELF LIFE

Stored at room temperature: 12 months

If to be stored for long periods, store in the refrigerator.

STORAGE RECOMMENDATIONS

Open package only when ready for use.

Use as basal dose.

Store in a cool, dry place out of direct sunlight.

Use before expiration date.

Store between 40 and 800 F

NATURE OF THE COMPOSITION OF THE PRODUCT:

Beneficial naturally occurring, GRAS, non GMO soil probiotics

Mobilizers of P, K, Mn, Zn etc. borne microorganisms

Natural carbon as non GM Corn based dextose as microbial Food

Nitrogen fixers

Organic Acid producing microbes

Enzyme secreting microbes

Amino acid producing microbes

MINIMUM ORGANISM CONTENT GUARANTEE:

No. Scientific Name Form Content (cfu/g) Sub-category

1 Azospirillum lipoferum Water Soluble Powder 1x108 Non Symbiotic Nitrogen fixing bacteria

2 Azotobacter chroococcum Water Soluble Powder 2x108 Non Symbiotic Nitrogen fixing bacteria

3 Rhizobium japonicum Water Soluble Powder 1x108 Symbiotic Nitrogen fixing bacteria

4 Peaenibacillus azotofixans Water Soluble Powder 1x108 Nitrogen fixing bacteria

5 Bacillus megaterium Water Soluble Powder 2x108 Phosphorus solubilizing bacteria

6 Bacillus polymyxa Water Soluble Powder 1x108 Nitrogen fixing bacteria

7 Pseudomonas striata Water Soluble Powder 1x108 Nitrogen fixing bacteria

8 Frateuria aurantia Water Soluble Powder 1x108 Potash mobilizing bacteria

9 Paenibacillus mucilaginosus Water Soluble Powder 1x108 Nitrogen fixing bacteria

10 Thiobacillus ferrooxidans Water Soluble Powder 1x106 Sulphur mobilizing bacteria

11 Bacillus globisporus Water Soluble Powder 1x106 Silica solublizing bacteria

12 Thiobacillus thiooxidans Water Soluble Powder 1x105 Iron, Zinc and Sulphur solublizing bacteria

13 Thiobacillus novellus Water Soluble Powder 1x105 Supplies Sulphur, Ferrous mobilizing bacteria

14 Corynebacterium spp Water Soluble Powder 1x105 Aminoacid producing Bacteria

15 Penicillium citrinum Water Soluble Powder 1x105 Manganese solubilizing bacteria

16 Willopsis saturnus Water Soluble Powder 1x105 Plant-growth-promoter

17 Glomus mosseae Water Soluble Powder 300 IP/gm Improves shoot length and weight

18 Trichoderma Harzianum Water Soluble Powder 1x106 Bio fungicide

19 Pseudomonas fluorescence Water Soluble Powder 1x106 Plant growth promoting rhizo bacteria. a

widespread facultative parasite of soil borne

pathogenic bacteria, pathogenic fungi and plant

parasitic nematodes

PLANT & ENVIRONMENTAL SAFETY

BIO SOIL MAGIC is totally harmless to plants even when recommended rates are exceeded.

BIO SOIL MAGIC is totally harmless to both humans and wildlife and is environment friendly, when used as

per recommendations.

USAGE SAFETY PRECAUTIONS

Keep out of reach of children and pets.

Use dust mask, safety glasses and protective gloves when applying BIO MAGIC.

Avoid prolonged or repeated skin contact and inhalation.

Please seek immediate medical attention in case of accidental ingestion.

LIMITED LIABILITY AND WARRANTY

Producer guarantees this product to produce satisfactory root formation and / or nitrogen fixation and / or

phosphorus mobilization under favorable soil and weather conditions when applied under the manufacturer’s

specifications before expiration date, or purchase price will be refunded.

Producer assumes no responsibility for loss or partial loss of crop from any cause whatsoever.

This Limited Warranty is in lieu of all other warranties, expressed or implied. The Limited Warranty is void

where prohibited by law. Producer must be notified of any field complaint in writing within forty five (45) days

after using the product. Many factors other than the above claims affect crop performance like: use of chemicals,

fungicides, pesticides, etc, which may affect the performance of this product in addition to weather conditions.

Organic farmland by world region (2000-2008)

UNIQUE CHARACTERISTICS OF BIO MAGIC

SYMBIOTIC NATURE OF THE PLANTS

It is very interesting to note that most of the plants feed by releasing root exudates of precise chemical

composition to activate their friendly soil fungi and bacteria which will solubilize elements required by the plant

at that time.

The exudate composition varies throughout the life of the plant, and any stresses imposed upon it result in further

compensatory changes - in essence, the plant practises self medication.

The term 'nature's smorgasbord' was coined to explain this process.

Biosafe

Consumer friendly

Creates non ambient conditions to the pests

Eco friendly

Economical

Ensures better life to the consumer of the produce

GRAS

Improved colour, texture, aroma, taste and shelf life of the

produce.

Helps in improving the quality of yield.

Increased nutritive profile of the yield

Increased quantity of produce

Assists in increasing the output

Latest technology

Makes the plant to grow stronger.

New and novel approach

Non toxic

Practical

Helps in preventing the crop diseases

Promotes plant burgeon earlier

Reduced plant protection costs

Simple

Stable

Strengthens the strength of growing

Unique concept

Mycorrhiza

'Nature's smorgasbord' provides a possible explanation for the prevalence of pest and disease attack in crops

fertilized by chemical means - applied soluble fertilizer masks the 'smorgasbord' process, eliminating correct

nutrition.

In this scenario, a novel concept comprises single dose basal application of BIO MAGIC, which is a total

product designed to provide a total nutrition to the plants throughout their life cycle; without the disadvantages of

Chemical fertilizers.

ROLE OF SOIL PROBIOTICS

Soil Probiotics are to help in

Absorption of minerals

Biodegradation of the residual pesticides in the Soil.

Ferments the organic matter

Bioremediation of the contaminated soil.

Fixing of Nitrogen

Redressal of the SOIL.

Solubilization of Nutrients like P, K, Iron, Sulfur, Mn, Zn

Supporting soil homeostasis

Maintain ambient pH

Degrades the pollutants

PGPSB (Plant Growth Promoting Soil Bacteria) promote growth of plants by one or more of various mechanisms:

colonizing roots to deliver biological fungicides and or pesticides to the plant when required

assisting the plants to attain abilities to improved tolerance to the menace of pests and insects

degrading the toxic materials secreted by plants and that are present in the rhizosphere

fermenting crude non absorbable plant residues etc., to convert into Total Digetable Nutrients (TDN).

helping the plants to attain resistance to biotic and abiotic stresses

lowering the level of stress ethylene induced by heavy metals such as nickel

producing siderophores which can solubilize and sequester iron from the soil and provide it to the plant;

providing fixed nitrogen and inducing synthesis of plant hormones

releasing enzymes necessary for solubilizing and mobilizing vital nutrients

supplying required phyto-hormones such as auxins, gibberellins and cytokinins

suppressing phyto pathogenic microorganisms;

MECHANISM OF ACTION

CITATIONS:

1. On nutrient basis 1 tonne of Rhizobium Biofertilizers is equivalent to 100 tonne of Fertilizer nitrogen (Verma

&Bhattacharyya,1991).

2. The utilization of biofertlizers can decrease the use of urea-N, prevent the depletion of soil organic matter and

reduce environmental pollution to a considerable extent.

► Cyanobacteria and PGPR are excellent model systems which can provide the biotechnologist with novel

genetic constituents and bioactive compounds having diverse uses in agriculture and environmental

sustainability.

Assisting in: Better drought tolerance

Better heat tolerance

Better Root development

Better root expansion

Enhanced growth rates

Improved energy levels in the soil.

Increased germination

Increased nutrient levels

Increased production

inhibition of pathogenic Fungus/Yeast/Mold

Repellence to pathogenic insects and pests

Resulting in yield with higher levels of Total Digestible Nutrients

► PGPR and cyanobacteria offer an environmentally sustainable approach to increase crop production and soil

health. (http://www.sciencedirect.com/science/article/pii/S0167880911000351)

3. As will be discussed later, crop growth and development are closely related to the nature of the soil microflora,

especially those in close proximity to plant roots, i.e., the rhizosphere.

Thus, it will be difficult to overcome the limitations of conventional agricultural technologies without

controlling soil microorganisms.

This particular tenet is further reinforced because the evolution of most forms of life on earth and their

environments are sustained by microorganisms.

Most biological activities are influenced by the state of these invisible, minuscule units of life.

Therefore, to significantly increase food production, it is essential to develop crop cultivars with

improved genetic capabilities (i.e., greater yield potential, disease resistance, and nutritional quality) and

with a higher level of environmental competitiveness, particularly under stress conditions (i.e., low

rainfall, high temperatures, nutrient deficiencies, and agressive weed growth).

To enhance the concept of controlling and utilizing beneficial microorganisms for crop production and

protection, one must harmoniously integrate the essential components for plant growth and yield

including light (intensity, photoperiodicity and quality), carbon dioxide, water, nutrients (organic-

inorganic) soil type, and the soil microflora.

Because of these vital interrelationships, it is possible to envision a new technology and a more energy-

efficient system of biological production.

Low agricultural production efficiency is closely related to a poor coordination of energy conversion

which, in turn, is influenced by crop physiological factors, the environment, and other biological factors

including soil microorganisms.

The soil and rhizosphere microflora can accelerate the growth of plants and enhance their resistance to

disease and harmful insects by producing bioactive substances.

These microorganisms maintain the growth environment of plants, and may have secondary effects on

crop quality.

A wide range of results are possible depending on their predominance and activities at any one time.

Nevertheless, there is a growing consensus that it is possible to attain maximum economic crop yields of

high quality, at higher net returns, without the application of chemical fertilizers and pesticides.

Until recently, this was not thought to be a very likely possibility using conventional agricultural

methods.

However, it is important to recognize that the best soil and crop management practices to achieve a more

sustainable agriculture will also enhance the growth, numbers and activities of beneficial soil

microorganisms that, in turn, can improve the growth, yield and quality of crops (National Academy of

Sciences, 1989; Hornick, 1992; Parr et al., 1992). (http://www.agriton.nl/higa.html)

4. Soil biota perform functions vital to the environment and, particularly, to agriculture.

They range from the regulation of soil structure and groundwater regimes to degradation of pollutants,

nutrient cycling, carbon sequestration, plant protection and growth enhancement, and ecosystem

purification.

No less important to plant productivity are microorganisms, the most abundant of all soil biota and

responsible for driving nutrient and organic matter cycling, soil fertility, soil restoration, and plant health

and production.

More than 90% of the world's plants develop symbiotic association with one of the five main types of

Mycorrhizae, a fungus that acts as a natural extension of the plant's root system.

This association increases the plants' capacity to take up nutrients, protects them against pathogens, and

increases their tolerance to pollutants and to adverse soil conditions, such as water deficiency, low pH

and high soil temperature.

Use of diazotrophic and endophytic associative bacteria that not only fix atmospheric nitrogen but

modify the shape and increase the number of root hairs, helping plants take up more nutrients.

The application of these organisms in inoculants (mostly in maize, rice, wheat and sugar cane) has led to

yield increases ranging from "negligible to almost 100%".

(http://www.fao.org/ag/magazine/0011sp1.htm)

5. Plant-growth promoting bacteria (PGPB) provide the host with essential services such as nitrogen fixation,

solubilization of minerals such as phosphorus, synthesis of plant hormones, direct enhancement of mineral

uptake, and protection from pathogens.

http://www.sciencedirect.com/science/article/pii/S0167880911000351

http://www.agriton.nl/higa.html

http://www.fao.org/ag/magazine/0011sp1.htm

(Kloepper, J. W (1993). "Plant growth-promoting rhizobacteria as biological control agents". In Metting, F. B.,

Jr. Soil microbial ecology: applications in agricultural and environmental management. New York: Marcel

Dekker Inc. pp. 255–274.)

6. PGPBs may protect plants from pathogens by competing with the pathogen for an ecological niche or a substrate,

producing inhibitory allelochemicals, or inducing systemic resistance in host plants to the pathogen.

(Compant S, Duffy B, Nowak J, Clément C, Barka EA (2005). "Use of Plant Growth-Promoting Bacteria for

Biocontrol of Plant Diseases: Principles, Mechanisms of Action, and Future Prospects". Appl Environ Microbiol.

71 (9): 4951–9.)

1. Azospirillum lipoferum

Common name

Nitrogen fixing bacteria

Azospirillum species are described as Gram negative, rod-shaped, 1mm in diameter, very motile.

Mode of action

Azospirillum has two different phases of attachment to wheat roots.

The primary adsorption phase is fast (reaches a maximum within 2 h of incubation), weak, and governed

probably by bacterial proteins.

The second phase (called anchoring) takes longer (it begins only after 8 h of incubation and reaches a maximum

after 16 h), is stronger and irreversible, and probably is based on bacterial extracellular surface polysaccharides

(Skvortsov et al. 1995; Del Gallo and Haegi 1990; Michiels et al. 1990; Zaady and Okon 1990).

The adsorption and anchoring are probably different phenomena.

Nitrogen fixation was the first mechanism proposed to explain improved plant growth following inoculation with

Azospirillum.

This was mainly because of an increase in the number of nitrogenous compounds and the nitrogenase activity in

inoculated plants.

Nitrite, either added directly or excreted by Azospirillum in nitrate respiration, similarly causes a sharp increase

in the formation of lateral roots.

It is possible that the growth promotion effect on wheat roots by Azospirillum is because of formation of nitrite

(Bothe et al. 1992).

Enhanced mineral uptake in the plant as a result of Azospirillum inoculation was a popular explanation for the

inoculation effects in the 1980s (for a review see Bashan and Levanony 1990).

Azospirillum can produce in vitro the phytohormones IAA, gibberellins, cytokinins (losipenko and Ignatov 1995;

Patten and Glick 1996; Rademacher 1994; for earlier literature, see Bashan and Levanony 1990), and ethylene

(Strzelczyk et al. 1994).

Whatever the exact mechanism, the fact that Azospirillum affects plant cell metabolism from outside the cell

(without entering the intact plant cells) suggests that the bacteria are capable of excreting and transmitting a

signal(s) which crosses the plant cell wall and is recognized by the plant membranes.

This interaction initiates a chain of events resulting in the observed altered metabolism of the inoculated plant.

Since plant membranes are extremely sensitive to any change, their response may serve as a precise indicator for

Azospirillum activity at the cellular level.

Although the nature of the released signal molecule is as yet unknown, it is proposed that plant membranes are

probably a primary target for Azospirillum on plant roots.

Scope of application

A possible agricultural breakthrough might be the use of Azospirillum as a helper bacterium together with other

beneficial microorganisms.

Azospirillum is not yet known as a biocontrol agent of soil borne plant pathogens.

Azospirillum lipoferum M produced catecholtype siderophores under iron-starved conditions that exhibited

antimicrobial activity against various bacterial and fungal isolates (Shah et al. 1992).

Azospirillum lipoferum was capable of reducing 4- chloronitrobenzene, an aromatic compound used in the

manufacturing of pesticides, dyes, explosives, and industrial solvents and an environmental pollutant (Russel and

Muszynski 1995).

2. Azotobacter chroococcum

Benefits the next crop also due to its residual effect.

Encourages better root development

Enhances soil health and soil fertility

Secretes growth hormones to increase crop productivity

Synthesizes biologically active substances like vitamins, nicotinic acid, indole acetic acid, gibberellins etc and

helps in better germination and good growth of the crop.

Azotobacter is free-living, nitrogen-fixing bacteria and is known to produce several plant growth promoting

subustances.

In addition to nitrogen fixation by these bacteria, they are also known to protect plants against pathogenic

microorganisims either by discouraging their growth or by destroying them

The range of nitrogen fixed by per ha/year varies from crop to crop; it is 80 - 85 kg for cow pea, 50 - 60 kg for

groundnut, 60 - 80 kg for soybean and 50 - 55 kg for moong bean.

Biological Particularity

Mode of action

Azotobacter naturally fixes atmospheric nitrogen in the rhizosphere.

There are different strains of Azotobacter each has varied chemical, biological and other characters.

However, some strains have higher nitrogen fixing ability than others.

Azotobacter uses carbon for its metabolism from simple or compound substances of carbonaceous in nature.

Besides carbon, Azotobacter also requires calcium for nitrogen fixation.

Similarly, a medium used for growth of Azotobacter is required to have presence of organic nitrogen, micro-

nutrients and salt in order to enhance the nitrogen fixing ability of Azotobacter.

Another individualistic trait of Azotobacter is their ability to synthesize not just one, but three nitrogenases.

The enzyme diversity and an extremely rapid metabolic rate (the highest of any known living organism) allow

the bacterium to fix nitrogen when oxygen is present.

Besides, nitrogen fixation, Azotobacter also produces Thiamin, Riboflavin, Nicotine, indole acetic acid and

gibberellins.

When Azotobacter is applied to seeds, seed germination is improved to a considerable extent, so also it controls

plant diseases due to above substances produced by Azotobacter.

Helps for better crop growth and seedling establishment.

Increase crop yield over 25%

Increases soil microbial activity

Increases germination of seeds

Enhances overall soil fertility

Produces various growth promoting substances, which ultimately increase seed germination, plant stand and pant

vigor.


The species of Azotobacter are known to fix on an average 10 mg.of N/g of sugar in pure culture on a nitrogen free

medium.

A maximum of 30 mg.N fixed per gram of sugar was reported by Lopatina.

Achieves about 10 to 15% increase of crop yield.

Azotobacter is tolerant to high salts.

Azotobacter is heaviest breathing organism and requires a large amount of organic carbon for its growth.

Azotobacter is less effective in soils with poor organic matter content.


Encourages better root development

Enhances soil health and soil fertility

Exhibit anti-fungal activities and thereby control fungal diseases caused by Alternaria, Fusarium and

Helminthosporium indirectly.

Fixes about 15 – 20 Kg. Atmospheric nitrogen per hectare when applied @ 4 x 109 CFU per Ha

Increases germination of seeds by about 15-30%.

It can benefit crops by Nitrogen fixation, growth promoting substances, fungi static substances.

It improves seed germination and plant growth

It is poor competitor for nutrients in soil

It thrives even in alkaline soils.

Produces Thiamin, Riboflavin, Nicotine.

Releases certain Growth Promoting Factors which are found very useful for increasing the seed germination,

plant growth and ultimately the yield.

Secretes growth hormones to increase crop productivity.

Synthesizes biologically active substances like indole acetic acid, gibberellins etc and helps in better germination

and good growth of the crop.

3. Rhizobium japonicum

Rhizobia can be found in the roots, or rhizosphere, of leguminous and other types of plants

They also have symbiotic relationships with legume plants, which can't live without these bacteria's essential

nitrogen-fixing processes.

In nodules, the rhizobia bacteriods use carbon and energy from the plant in the form of dicarboxylic acids.

Recent studies have suggested that the bacteroids do more than just provide the plant with ammonium (through

nitrogen fixation).

Rhizobium can use the amino acids from the plant to shut down their ammonium assimilation.

The bacteroids "act like plant organelles to cycle amino acids back to the plant for asparagine synthesis," making

the plant dependent on them (Lodwig et al. 2003).

This system creates mutualism between the bacteria and the plant.

The bacteria colonize plant cells within root nodules; here the bacteria converts atmospheric nitrogen to

ammonia and then provides organic nitrogenous compounds such as glutamine or ureides to the plant.

The plant provides the bacteria organic compounds made by photosynthesis.

Process of nitrogenase-dependent hydrogen production is a major factor in the efficiency of symbiotic nitrogen

fixation.

To have more efficient energy use, some Rhizobium and many Bradyrhizobium strains recycle the hydrogen

produced by nitrogenase in nodule bacteroids that have a hydrogen uptake system (Hup).

It is known that the rhizobia gene nodZ play a role in fucosylating the lipochitin oligosaccharide (LCO) signals

molecules.

NodZ proteins catalyze the transfer of an α-L-fucopyranosyl residue from GDP-β-L-Fucose (GDP-Fuc) to the C-

6 position of the GlcNAc at the reducing end of the nodulation (Nod) factors.[

Quantity of biological N fixed by Liqiud Rhizobium in different crops

Host Group Rhizobium Species Crops N fix kg/ha

Pea group Rhizobium leguminosarum Green pea, Lentil 62- 132

Soybean group R.japonicum Soybean 57- 105

Lupini Group R. lupine orinthopus Lupinus 70- 90

Alfafa grp.Group R.mellilotiMedicago Trigonella Melilotus 100- 150

Beans group R. phaseoli Phaseoli 80- 110

Clover group R. trifoli Trifolium 130

Cowpea group R. species Moong, Redgram,

Cowpea, Groundnut

57- 105

Cicer group R. species Bengal gram 75- 117

Allen (1958) has listed four indicators that, if positive, the inoculation would be beneficial:

1. The absence of the same or symbiotically-related legume in the immediate past history of the land

2. Poor nodulation when the same crop was grown on the land previously

3. When the legume followed a non-legume in the rotation

4. When the land was undergoing reclamation

Rhizobial inoculant is often exposed to unfavorable environmental factors when inoculated seed are planted in

the soil.

These environmental factors are salt and osmotic stresses, soil moisture deficiency, high temperature and heat

stress, soil acidity and alkalinity, and nutrient deficiency stress etc (Zahran, 1999; Hungria and Vargas, 2000).

Water-soluble substances from the seed coat may be inhibitory to rhizobia.

Aluminium toxicity affects the growth of rhizobia (Cooper et al., 1983; Coventry and Evans, 1989; Campo,

1995), and symbiosis (Murphy et al., 1984; Brady et al., 1990; Campo, 1995).

One of the most popular hypotheses is that EPS help soil microorganisms to survive desiccation.

The Rhizobial cells are surrounded by a layer of exopolysaccharide (EPS), which has been reported to have a

role in protecting cells under stress conditions, such as exposure to toxic elements and desiccation by slowing the

rate of drying within colony microenvironment (Hatel and Alexander, 1986; Roberson and Firestone, 1992;

Ophir and Gutnick, 1994).

Some work has been studied the binding of acidic exopolysaccharide produced by Bradyrhizobium strain and

several metal cations (Corzo et al., 1994).

Bradyrhizobium EPS was precipitated by Fe3+, Tn4+, Sn2+, Mn2+, Co2+ and Al3+.

The presence of Fe3+ increased the EPS precipitation by aluminum.

Aluminium has been reported to be toxic for both the members of the family Rhizobiaceae and the plants.

So, the complexation and precipitation of Al3+ by EPS could be a detoxifying mechanism that promotes survival

of rhizobia in high metallic cations soil.

RHIZOBIUM'S INTERACTION WITH LEGUME PLANTS

1. Multiplication of rhizobia at or near the root surface

2. Adhesion of rhizobia to root hair surface

3. Root hair branching and root curling

4. Formation of an infection thread

5. Nodule initiation: formation of nodule meristem, nodule development and differentiation

6. Bacteroid release from infection thread

7. Bacterial differentiation

8. Onset of nitrogen fixation

9. Biochemcial and physiological functions associated with nitrogen fixation

10. Maintenance (persistence) of nodule function

11. Boonkerd (2002) investigated the soybean productivity and economic gain due to different cultural practices and

observed more than 50% increase in soybean yield due to Bradyrhizobium application.

It was found in his experiment that 1,250 g of inoculant could replace 179.2 kg of urea fertilizer, or 200 g of

Bradyrhizobium inoculant for 28.6 kg of urea.

12. Symbiotic nitrogen fixation is an important source of nitrogen, and various legume crops could fix as much as

200 to 300 kg of nitrogen per hectare or about 70 million metric tons of nitrogen per year (Peoples et al., 1995;

Brockwell et al., 1995).

13. Therefore, legume inoculation with rhizobial inoculant is one way to ensure that specific rhizobial strain for that

host plant is present in the soil at the proper time and in sufficient number to assure a quick and effective

nodulation and efficient subsequent nitrogen fixation (Cleyet-Marel, 1988).

14. Rhizobium previously well known as a symbiotic N fixer is reported as asymbiotic (associative & endophytic)

microorganisms in recent years (Biswas et al., 2000a, b).

The most inoculation studies have focused on free living diazotrophs, although a few reports indicate rhizobia

can act as plant growth promoting rhizobacteria (PGPR) (Hoflich et al., 1995; Noel et al., 1996; Yanni et al.,

1997).

The PGPR influence the crop growth and development by releasing plant growth regulators (Glick & Bashan,

1997; Volpin & Philips, 1998) and improving morphological characteristics uptake (Okon & Kapulnik, 1986).

The growth promoting effects of rhizobacteria may include phytohormone production (Chabot et al., 1996), N2

fixation (Urquiaga et al., 1992) and more efficient use of nutrients (Chabot et al., (1996). Yanni et al. (1997) and

Biswas (1998) reported increased N uptake in rice plants inoculated with rhizobia.

4. Peaenibacillus azotofixans Synonyms:

Paenibacillus durus (Smith and Cato 1974) Collins, Lawson, Willems, Cordoba, Fernandez-Garayzabal, Garcia, Cai,

Hippe and Farrow 1994 VP

Bacillus azotofixans Seldin, Van Elsas and Penido 1984 VP

Paenibacillus azotofixans (Seldin, Van Elzas and Penido 1984) Ash, Priest and Collins 1994 VL

Objective synonyms:

Clostridium durum Smith and Cato 1974 AL

4. Paenibacillus azotofixans

Synonyms:

Bacillus azotofixans Seldin et al.

Strain Designations P3L-5

Isolation Wheat roots, Parana State, Brazil

Type Strain yes

Biosafety Level 1

Medium Medium 18: Trypticase Soy Agar/Broth

Growth Conditions

Temperature: 30.0°C

Paenibacillus azotofixans is a nitrogen-fixing bacterium often found in soil and in the rhizospheres of different

grasses.

Strains belonging to the species Paenibacillus azotofixans were shown to be efficient nitrogen fixers prevalent in

the rhizospheres of maize, sorghum, sugarcane, wheat, banana, and forage grasses.

Some strains are able to produce antimicrobial substances and solubilize organic phosphates

(Rosado A S, de Azevedo F S, da Cruz D W, van Elsas J D, Seldin L. Phenotypic and genetic diversity

of Paenibacillus azotofixans strains isolated from the rhizoplane or rhizosphere soil of different grasses. J Appl

Microbiol. 1998;84:216–226.).

Identification and maintenance of P. azotofixans isolates.

All presumptive P. azotofixans strains (white, convex, and mucous colonies isolated in thiamine-biotin solid

medium, after incubation under anaerobic conditions) were identified by the biochemical tests proposed by

Gordon et al.

(Gordon R E, Haynes W C, Pang H-N. The genus Bacillus. Agriculture handbook 427. Washington, D.C:

Agricultural Research Service, U.S. Department of Agriculture; 1973.).

Cellular morphology, forms and positions of spores, and swelling of the sporangia were observed by microscopy

of crystal violet-stained smears. Three other carbohydrates (sorbitol, dulcitol, and starch) were used to group the

strains into one of five groups based on fermentation patterns described previously

(Rosado A S, de Azevedo F S, da Cruz D W, van Elsas J D, Seldin L. Phenotypic and genetic diversity

of Paenibacillus azotofixans strains isolated from the rhizoplane or rhizosphere soil of different grasses. J Appl

Microbiol. 1998;84:216–226.; Seldin L, Penido E G C. Identification of Bacillus azotofixansusing API

tests. Antonie Leeuwenhoek. 1986;52:403–409.).

Whenever necessary, the API 50CH microtube system (bioMérieux, Marcy l’Etoile, France) was used in addition

to conventional tests. The API test galleries were prepared and read as described in the work of Seldin and

Penido.

In addition to these tests, a molecular method for identification of P. azotofixans was used. It consisted of PCR

amplification of part of the variable regions V1 to V4 of the 16S rRNA gene with two primers, which were

BAZO1 and BAZO2, followed by hybridization with a specific 18-bp oligonucleotide probe homologous to part of

the intervening region. The PCR product generated with P. azotofixans strains was 565 bp long and was

specifically detected by a P. azotofixans-specific probe, BAZOP

(Rosado A S, Seldin L, Wolters A C, van Elsas J D. Quantitative 16S rDNA-targeted polymerase chain reaction

and oligonucleotide hybridization for the detection of Paenibacillus azotofixans in soil and the wheat

rhizosphere. FEMS Microbiol Ecol. 1996;19:153–164.).

Strains identified as P. azotofixans were stored aerobically at room temperature on GB agar slants supplemented

with 1% CaCO3 (wt/vol).

Nitrogen fixation.

Acetylene reduction was measured by assessing the ethylene production of cultures in 18-ml vials as described

previously

(Seldin L, van Elsas J D, Penido E G C. Bacillus azotofixans sp. nov., a nitrogen-fixing species from brazilian soils

and grass roots.Int J Syst Bacteriol. 1984;34:451–456.).

Solubilization of organic phosphate.

Aliquots (10 μl) of young cultures of P. azotofixans were spot inoculated onto calcium phytate agar plates and

incubated for 5 days at 30°C. A positive result was considered the formation of a clear zone around the growth

spot.

Detection of inhibitory substances.

The activities of P. azotofixansstrains inhibiting a strain of Corynebacterium fimi were assayed by the lawn-

spotting technique described by Seldin and Penido

(Seldin L, Penido E G C. Production of a bacteriophage, a phage tail-like bacteriocin and an antibiotic by Bacillus

azotofixans. An Acad Bras Cienc. 1990;62:85–94.).

Resistance to heavy metals and to antibiotics.

Resistance to the heavy metals and antibiotics listed here was determined by spot inoculating 18-h-old cultures

of P. azotofixans on TBN agar plates supplemented with CuSO4 (250 μg/ml), NiSO4 (263 μg/ml), HgCl2 (20 μg/ml),

CoSO4 (240 μg/ml), chloramphenicol (10 μg/ml), erythromycin (10 μg/ml), or rifampin (10 μg/ml).

REFERENCES:

1. Seldin L, et al. Bacillus azotofixans sp. nov., a nitrogen-fixing species from Brazilian soils and grass roots.

Int. J. Syst. Bacteriol. 34: 451-456, 1984.

2. Validation list no. 52. Int. J. Syst. Bacteriol. 45: 197-198, 1995.

3. Ash C, et al. Molecular identification of rRNA group 3 bacilli (Ash, Farrow, Wallbanks and Collins) using

a PCR probe test. Antonie van Leeuwenhoek 64: 253-260, 1993. PubMed: 8085788

4. Rosado AS, et al. Reclassification of Paenibacillus durum (formerly Clostridium durum Smith and Cato

1974) Collins et al. 1994 as a member of the species P. azotofixans (formerly Bacillus azotofixans Seldin et al.

1984) Ash et al. 1994. Int. J. Syst. Bacteriol. 47: 569-572, 1997.

5. De Vos P, Truper HG. Judicial Commission of the International Committee on Systematic Bacteriology

IXth International (IUMS) Congress of Bacteriology and Applied Microbiology. Minutes of the meetings, 14, 15

and 18 August 1999, Sydney, Australia. Int. J. Syst. Evol. Microbiol. 50: 2239-2244, 2000.

References :

M. Boon, C. Ras, J. J. Heijnen; The ferrous iron oxidation kinetics of Thiobacillus ferrooxidans in batch

cultures; Applied Microbiology and Biotechnology; Issue: Volume 51, Number 6; June 1999; Pages: 813 – 819

Paul, E.A., Clark, F.E. 1996. Soil Microbiology and Biochemistry. Academic Press. San Diego, CA. 340 pp.

Chapelle, Francis H. 1993. Ground-Water Microbiology and Geochemistry. John Wiley & Sons, Inc. New York,

NY. Pp 98-99.

Atlas, Ronald M., Parks, Lawrence C. 1993. Handbook of Microbiological Media. CRC Press. Boca Raton, FL.

1079 pp.

Holt, J.G., N.R. Krieg, P.H.A. Sneath, J.T. Staley, and S.T. Williams. 1994. Bergy’s Manual of Determinative

Microbiology. Ninth Ed. Williams and Wilkins. Baltimore, MD. 787 pp.

S. Malhotra, A. S. Tankhiwale, A. S. Rajvaidya and R. A. Pandey ; Optimal conditions for bio-oxidation of

ferrous ions to ferric ions using Thiobacillus ferrooxidans; Bioresource Technology, Volume 85, Issue 3,

December 2002, Pages 225-234

http://www.asm.usa.org/

http://commtechlab.msu.edu/sites/dlc-me/

5 Bacillus megaterium

Biological Particularity

Mode of action

The bacteria are capable of stimulating plant growth by releasing nutrients and growth promoters in the soil.

The AI of P Sol B, B. megaterium on reaching the soil becomes activated and produces fresh batch of live cells,

which grow and multiply, utilizing the carbon sources from the soil or from the root eudates.

During their growth they solubilise the fixed Phosphorous and various nutrient metals in the soil and make them

available to the plant in an easily assimible form.

Plant-growth promotion elicited by B. megaterium in Arabidopsis correlates with dramatic changes in root-

system architecture that include increased growth of lateral roots and increased root-hair elongation.

Detailed cellular measurements and CycB1,1:uidA expression analyses in the root meristem suggest that bacterial

inoculation inhibits both meristematic activity and cell elongation in the primary root and promotes epidermal-

cell differentiation.

The involvement of auxin and ethylene in mediating the observed developmental alterations was tested using the

auxin-responsive marker DR5:uidA and auxin- and ethylene-related mutants.

B. megaterium inoculation inhibits meristematic activity and reduces cell elongation in primary roots of

Arabidopsis.

These results suggest that a diffusible bacterial metabolite, possibly a plant growth–regulating substance caused

the described alterations on growth and development.

These results suggest that attenuation of meristematic activity in the primary root may be sufficient to elicit

major developmental alterations in the whole root system, including the premature differentiation of root hairs

and maturation of lateral roots.

This result also suggests that one or more of the metabolites produced by B. megaterium likely acts upstream of

rhd6 in the signaling cascade that leads to alterations in root-hair development.

Whether acetoin production by B. megaterium accounts for growth and developmental responses elicited in

Arabidopsis remains to be determined.

(José López-Bucio, Juan Carlos Campos-Cuevas, Erasto Hernández-Calderón, Crisanto Velásquez-

http://www.sciencedirect.com/science?_ob=GatewayURL&_method=citationSearch&_uoikey=B6V24-46M1HMG-2&_origin=SDEMFRHTML&_version=1&md5=1c89c1d0ea6f6d504735166b65523aa9

http://www.sciencedirect.com/science?_ob=GatewayURL&_method=citationSearch&_uoikey=B6V24-46M1HMG-2&_origin=SDEMFRHTML&_version=1&md5=1c89c1d0ea6f6d504735166b65523aa9

http://www.asm.usa.org/

http://commtechlab.msu.edu/sites/dlc-me/

Becerra, Rodolfo Farías-Rodríguez, Lourdes Iveth Macías-Rodríguez, and Eduardo Valencia-Cantero; Bacillus

megaterium Rhizobacteria Promote Growth and Alter Root-System Architecture Through an Auxin- and

Ethylene-Independent Signaling Mechanism in Arabidopsis thaliana; The American Phytopathological Society

MPMI Vol. 20, No. 2, 2007, pp. 207–217.)


B. megaterium could be used as a biocontrol agent against postharvest fungal disease caused by A. flavus.

(http://130.88.242.202/medicine/Aspergillus/articlesoverflow/20156660.pdf)

Several studies show that Bacillus megaterium determines a good bio-solubilisation of metals

There are notable bio-solubilisations for manganese (80%) and iron (60%).

The Gram-positive bacterium Bacillus megaterium is frequently employed for the industrial production of

exoenzymes including amylases, proteinases, and penicillin acylases, for instance.

Amongst the Plant Growth Promoting Rhizobacteria (PGPR), Bacillus megaterium have been advocated as

effective and economical bio inoculant to use in the integrated nutrient and pest control system.

Several strains of B. megaterium, B. licheniformis, B. coagulans, B. circulans isolated from shrimp farm

environments showed anti-vibrio activity.

Treatment with B. megaterium resulted in a greater than 40% reduction in nematode penetration and gall

formation compared with non-treated rice roots, and, in a separate study, colonization of rice roots with B.

megaterium decreased migration of M. graminicola to the root zone by nearly 60% compared with that of non-

treated roots.

Exposure of M. graminicola eggs to secondary metabolites of B. megaterium reduced hatching by over 60%

compared with eggs not exposed to the bacteria (J.L. Padgham and R.A. Sikora; Biological control potential and

modes of action of Bacillus megaterium against Meloidogyne graminicola on rice; Crop Protection; Volume 26,

Issue 7, July 2007, Pages 971-977).

Encourages early root development

Enhances crop yield by 15-20%

Fixes 20 Kg P/Acre/Year

Helps in rapid cell development.

Produces organic acids like succinic acid which hastens the maturity and thereby increases the ratio of grain and

yield.

Reduces chemical P fertilizer usage by 30-40%.


Encourages early root development.

Enhances soil health and soil fertility.

Increases pest and disease resistance in crops.

Produces organic acids like malic, succinic, fumaric, citric, tartaric and alpha ketoglutaric acid which hastens the

maturity and increases the ratio of grain to straw as well as the total yield.

Secretes growth hormones to increase crop productivity.

Stimulates formation of fats, convertible starches and result in healthy seeds.

The release of inorganic phosphate from organic phosphates is called mineralization and is caused by

microorganisms breaking down organic compounds.

The activity of microorganisms is highly influenced by soil temperature and soil moisture.

The process is most rapid when soils are warm and moist but well drained.

By increasing soil microbial activities, bioavailability of P in a bioactive soil was remarkedly enhanced. (Thien

and Myers, 1992).

It is generally recognized that organic acids solubilize RP through protonation and / or chelation reactions (Sagoe

et al., 1998).

Filamentous fungi are widely used as producers of organic acids (Matty, 1992)

The ability of low molecular weight organic acids, to release P from ores or rocks, related to their ability to form

stable metal complexes is well established (Mattey, 1992).

The principal mechanism for organic phosphate solubilization is acid phosphatase activity (McGrath et al.,

1995).

Besides the acid strength, the type and position of the ligand determine the effectiveness of the organic acid in

the solubilization process (Kpomblekou and Tabatabai, 1994).

The principal underlying mechanism of action of chelators is formation of unionized association compounds

with Ca++, Fe++, Al+++ and thus, increasing soluble phosphate concentration by scavenging phosphate from

mineral phosphates.

Although higher concentrations of P were not effective for PS activity, growth, however, increased successively.

http://130.88.242.202/medicine/Aspergillus/articlesoverflow/20156660.pdf

http://www.sciencedirect.com/science/journal/02612194

http://www.sciencedirect.com/science?_ob=PublicationURL&_tockey=%23TOC%235011%232007%23999739992%23653362%23FLA%23&_cdi=5011&_pubType=J&view=c&_auth=y&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=107424adb08fcb42e363dcd87481579a

http://www.sciencedirect.com/science?_ob=PublicationURL&_tockey=%23TOC%235011%232007%23999739992%23653362%23FLA%23&_cdi=5011&_pubType=J&view=c&_auth=y&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=107424adb08fcb42e363dcd87481579a

The limiting level of P in most soils provides the ecophysiological basis for positioning associations between

plant roots and mineral phosphate solubilizing (MPS) and/or organic phosphate solubilizing microorganisms

(OPS).

These associations are assumed to play an important role in phosphorus nutrition in many natural and agro-

ecosystems.

Plant growth studies

Small plantlets are taken and in the given way they are allotted for different agroclimatic zones and one group

were allotted for keeping as a control. Soil, manure and sand was mixed in the ratio of 1:1:1/2 respectively.

This soil mixture was filled to 33 pots and sesame seeds were sowed to it. Pots were labeled as 1,2,3,… upto 10

and C (control).

This was done for 3 replicas.

When plants were grown for certain length, the bacterial suspenion of 15ml was inoculated for each plant.

These bacterial suspensions, was diluted to 1:4 ratio, before inoculating into plants.

For control only water was added.

Observation for Plant growth

After 10 days, 25 days and 40 days the length of plants, number of leaves, number of branches and root length with the

dry weight of shoot and root system.

Seed germination studies Treatment allocation:

For this also, the same methodology is done for inoculation.

Tc - Control (uninoculated control),

T1 –from Zone 1,

T2 - from Zone 2 ,

T3 - from Zone 3 ,T4 - from Zone 4 ,T5 - from Zone 5 ,T6 - from Zone 6 ,T7 - from Zone 7 ,T8 - from Zone 8,T9- from

Zone 9 ,T10- Zone 10 .

Here zone refer to Agro climatic zone.

Seed bacterization

Two weeks old seeds were soaked in sterilized distilled water for overnight.

Then the seeds were dried and transfered in to respective zone cultures for 30 to 45 min.

After the respective zone soaked seeds were transfered into respective petriplates, they were covered with

Wattmen No.1 filter paper.

The plates were wetted using sterilized water.

After 3 days incubation, the length of the radical and plumule was recorded.

RESULTS

Table1: Growth of shoots in various zones.

S.No. Agro climatic

zone

Length of root

part in cm

Length of shoot

part in cm

The complete

length in cm 1 Control

2 Z-1

3 Z-2

4 Z-3

5 Z-4

6 Z-5

7 Z-6

8 Z-7

9 Z-8

10 Z-9

11 Z-10

Effect of plant growth on Sesamum indicum on both roots and shoots.

6. Bacillus polymyxa

Paenibacillus polymyxa is a plant growth-promoting rhizobacterium with a broad host range, but so far the use of

this organism as a biocontrol agent has not been very efficient.

In previous work we showed that this bacterium protects Arabidopsis thaliana against pathogens and abiotic

stress (S. Timmusk and E. G. H. Wagner, Mol. Plant-Microbe Interact. 12:951-959, 1999; S. Timmusk, P. van

West, N. A. R. Gow, and E. G. H. Wagner, p. 1-28, in Mechanism of action of the plant growth promoting

bacterium Paenibacillus polymyxa, 2003).

Here, we studied colonization of plant roots by a natural isolate of P. polymyxa which had been tagged with a

plasmid-borne gfp gene.

Fluorescence microscopy and electron scanning microscopy indicated that the bacteria colonized predominantly

the root tip, where they formed biofilms.

Accumulation of bacteria was observed in the intercellular spaces outside the vascular cylinder.

Systemic spreading did not occur, as indicated by the absence of bacteria in aerial tissues.

Studies were performed in both a gnotobiotic system and a soil system.

The fact that similar observations were made in both systems suggests that colonization by this bacterium can be

studied in a more defined system.

Problems associated with green fluorescent protein tagging of natural isolates and deleterious effects of the plant

growth-promoting bacteria are discussed.

We report here that P. polymyxa forms biofilms around the root tip and behaves as a root-invading bacterium and

as a DRB under particular experimental conditions.

As expected, due to the different environmental conditions, the population densities of both rhizosphere-

colonizing and root-invading bacteria differ in the gnotobiotic and soil systems (Fig. (Fig.5).5).

These results confirm that colonization by the bacterium can be modeled in the gnotobiotic system and that more

detailed studies on biofilm formation can be performed using this model.

Based on this study, niche exclusion and mechanical protection of the nonsuberized regions of roots will be

pursued as a possible mechanism of P. polymyxa biocontrol and drought tolerance-enhancing activity.

(Salme Timmusk,* Nina Grantcharova, and E. Gerhart H. Wagner; Paenibacillus polymyxa Invades Plant Roots

and Forms Biofilms; Appl Environ Microbiol. 2005 Nov; 71(11): 7292–7300.)

7. Pseudomonas striata Pseudomonas striata Chester

1. Benefits the next crop also due to its residual effect.

2. Encourages early root development

3. Enhances soil health and soil fertility

4. Increases pest and disease resistance in crops

5. Produces organic acids like malic, succinic, fumaric, citric, tartaric and alpha ketoglutaric acid which hastens the

maturity and increases the ratio of grain to straw as well as the total yield.

6. Secretes growth hormones to increase crop productivity

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1287669/figure/f5/

7. Stimulates formation of fats, convertible starches and result in healthy seeds

Biochemical Characteristics of Pseudomonas striata Chester

Gram negative, 0.7-1.1 / 2-4 μm, motile rods with polar multitrichous flagella.

Recommended for all soils and crops.

8 Frateuria aurantia

Synonynms:

Acetobacter aurantium ex Kond and Ameyama

Frateuria aurantia produced yellow-coloured colonies, Gram-negative, straight rods, 0.5 to 0.7 by 0.7 to 3.5 µm,

which occur singly or in pairs, rarely in filaments or as irregular cells and never in chains.

Motile by means of polar flagella.

The available potassium in soil increases when mixed soluble mineral fertilizers (SS+KCl) are applied, probably

due to the higher concentration of potassium in the soluble mineral fertilizer (KCl), followed by the treatment

with potassium biofertilizer (K Sol B) in the rate K Sol B240.

It is important to know that there are not many references about application of potassium biofertilizers produced

from powdered rocks.

In soils of the coastal tableland of Pernambuco State, after sugar cane cultivation, Stamford et al. (2006)

described increasing availability of K in soil when K rock biofertilizer plus elemental sulfur inoculated with

Acidithiobacillus were applied, similar to the results found in the present study with melon.

Lima et al. (2007) verified positive and signifcant effect of P and K fertilization in available K in soil, after

lettuce crop in the region of Cariri, and the best results were obtained when KCl was applied at 160 kg ha-1

, and

with K rock biofertilizer (KB) in the rate KB80.

(http://www.scielo.br/scielo.php?pid=S0102-05362009000400008&script=sci_arttext)


Potassium is one of the three macronutrients for plants.

The potassium content of fertilizer is given in terms of K2O. It plays important role in plant physiology and

improves yield. India imports its entire need of approximately 1.2 million tonnes of potash fertiliser.

There is no economically viable source of conventional potash deposit in India.

In clay soils there are no leaching losses of potassium whereas, in sandy and organic soils such losses occur.

It is important that there should be continuous supply of potash from sowing until harvest.

Abundant K supply reduces bacterial population in general in root zone including denitrifying bacteria.

K-fertilisers should be applied after soil tests/plant analyses before any symptoms appear on plants.

In calcareous soils/recently limed soils having large number of calcium cations, higher level of K is required.

Plant roots absorb most of required potassium through soil moisture. Inadequate soil moisture may result poor

absorption of K-nutrient by plant.

Reduced soil temperature & liming results in low amount of potassium in soil solution.

Poor soil aeration also reduces potassium uptake by plants.

Conventional K-fertilisers are muriate of potash (KCl) and sulphate of potash (K2SO4).

Non-Conventional K-sources include:

(i) Glauconite a slow release fertiliser.

It is available in huge quantity in India.

Green sand containing large percentage of Glauconite has been used as potash in USA, England, France,

Belgium etc.

Glauconitic sand has also been beneficiated to produce commercial fertiliser.

Occurrence of these deposits very near to earth surface, add to economy.

These deposits containing 4 to 7% of K2O are comparable to New Jersey green sand (average K2O, 6%).

Glauconitic sand is suitable for application in acidic soils.

30% of the cultivated land in India is acidic.

(ii) The sludge/powder generated during polishing and cutting of granite contains K2O ranging 4-5% in addition

to other micro nutrients Mg, Ca, Fe etc. and can be used as K-fertiliser.

(iii) Potash salts obtained by beneficiation of marine bitterns.

About 7 million tonnes of salt is produced at Indian coast line.

As per CSMCRI 2.4 tonnes of potassium Schoenite (K2SO4.MgSO4.6H 2O) can be manufactured for every

100 tonnes of common salt.

Other potassium salts obtained from sea bittern include carnallite & Kainite.

http://www.scielo.br/scielo.php?pid=S0102-05362009000400008&script=sci_arttext

(iv) Potash salts produced as by product of aluminum production, cement production, sugar production

(molasses) etc.

(v) Fly ash from coal-burning power plants.

9. Paenibacillus mucilaginosus Synonyms:

Bacillus mucilaginosus

Bacillus mucilaginosus Avakyan et al. 1998 emend. Shelobolina et al. 1998

Paenibacillus mucilaginosus (Avakyan et al. 1998) Hu et al. 2010

CHARACTERISTICS

Paenibacillus is a genus of facultative anaerobic, endospore-forming bacteria, originally included within the

genus Bacillus and then reclassified as a separate genus in 1993.

Bacteria belonging to this genus have been detected in a variety of environments such as: soil, water,

rhizosphere, vegetable matter, forage and insect larvae, as well as clinical samples.

The name reflects this fact: Latin paene means almost, and so the Paenibacilli are literally almost Bacilli.

More specifically, several Paenibacillus species serve as efficient plant growth promoting rhizobacteria (PGPR).

PGPR competitively colonize plant roots and can simultaneously act as biofertilizers and as antagonists

(biopesticides) of recognized root pathogens, such as bacteria, fungi and nematodes.[22]

They enhance plant growth by several direct and indirect mechanisms.

Direct mechanisms include phosphate solubilization, nitrogen fixation, degradation of environmental pollutants

and hormone production. Indirect mechanisms include controlling phytopathogens by competing for resources

such as iron, amino acids and sugars, as well as by producing antibiotics or lytic enzymes.

Competition for iron also serves as a strong selective force determining the microbial population in the

rhizosphere.

Several studies show that PGPR exert their plant growth-promoting activity by depriving native microflora of

iron.

Although iron is abundant in nature, the extremely low solubility of Fe3+ at pH 7 means that most organisms

face the problem of obtaining enough iron from their environment.

To fulfill their requirements for iron, bacteria have developed several strategies, including:

I. The reduction of ferric to ferrous ions,

II. The secretion of high-affinity iron-chelating compounds, called siderophores, and

III. The uptake of heterologous siderophores.

P. vortex's genome for example, harbors many genes which are employed in these strategies, in

particular it has the potential to produce siderophores under iron limiting conditions.

Despite the increasing interest in Paenibacillus spp. genomic information of these bacteria is lacking.

More extensive genome sequencing could provide fundamental insights into pathways involved in complex social

behavior of bacteria, and can discover a rich source of genes with biotechnological potential.

APPLICATIONS

Paenibacillus mucilaginosus is a widely used biofertilizer, providing P, K and N nutrients.

CITATIONS:

1. Diversity of plant-growth-promoting Paenibacillus mucilaginosus isolated from vegetable fields in Zhejiang, China

Xue Aiqin, Cao Zhaoyun, Zhang Shanshan, Wu Duqing, Hu Xiufang

(Institute of Bioengineering, Zhejiang Sci-Tech University, HangZhou 310018)

Foundations: National High Technology Research and Development Program of China

Brief author introduction:Xue Aiqin，1986-），female，master，microbiology

Correspondance author: Hu xiufang (1971-)，female， researcher，microbiology. E-mail: [email protected]

Abstract: Paenibacillus mucilaginosus is a widely used biofertilizer, providing P, K and N nutrients.

In the present study, the diversity of 27 P. mucilaginosus strains in phenotype and genotype were investigated to

find out the principal factor influencing the functions.

The strains were characterized to show certain similar shared morphological and biochemical characteristics, yet

they showed diversity in sizes of capsules and colonies, productions of polysaccharides and acid, P, and K-

dissolution and N-fixation.

The sizes of the colonies and capsules varied from 2.00-5.72 mm and 200.07-857.23 μm2, respectively.

The polysaccharide production was 2.04-13.12 mg•ml-1, which was positively correlated with capsule (r=0.714,

P<0.05) and colony (r=0.824, P<0.05) sizes.

The strains caused pH decrease from 1.21 to 2.62, which was positively correlated with the dissolution of P

(r=0.777, P<0.05) and K (r=0.778, P<0.05).

The concentration rangess of dissolved P and K were 0.05-102.72 mg•l-1 and 0.02-27.92 mg•l-1, respectively.

The fixed total nitrogen was 0.47-18.28 mg•l-1.

The strains also showed genotypic diversity at gene, repetitive sequence and genome levels.

Altogether 249 amplified bands were obtained using 18 primers in RAPD pattern, and the dendrogram showed

that the strains were grouped into 2 clusters according to their origins.

Each cluster was divided into two branches: one was clustered as the strains with higher polysaccharide and acid

production, and the other was clustered as the strains with lower production.

The gyrB gene and ERIC-PCR gave the similar results.

In conclusion, as RAPD and gyrB gene showed good correspondence with polysaccharide and acid diversity,

they can serve as biomarkers and thus be employed efficiently in species identification and selection at a large

scale.

INTRODUCTION:

P. mucilaginosus is able to degrade insoluble soil minerals, with the release of potassium and water-soluble

phosphorous [2, 4-6], and some of the strains have antagonistic [7, 8] or plant growth-promoting [9] properties.

Therefore, P. mucilaginosus preparations have been successfully used as a biofertilizer in the planting of

tobacco, tomato, groundnut, and sudan grass [5, 10-12] since the 1990s.

Recently, P. mucilaginosus,which has the characteristic of secreting exocellular polysaccharides on a nitrogen-

free medium [13], has been shown to be an effective bioflocculant for flocculating inorganic and organic

suspended solids, and also absorbs heavy metals in wastewater [14, 15].

It plays a potential role in the treatment of wastewater and recovery of valuable metals [16, 17].

(http://www.google.co.in/url?sa=t&rct=j&q=&esrc=s&source=web&cd=2&ved=0CDwQFjAB&url=http%3A%2F%2F

www.paper.edu.cn%2Fen_releasepaper%2FdownPaper%2F201012-

617.html&ei=IugVUZHPCYiurAea4YGYAQ&usg=AFQjCNF3Ss2x6gRMUWo84TFH0uwdUnfVPw&bvm=bv.42080

656,d.bmk)

Paenibacillus mucilaginosus is critical silicate bacteria in the biogeochemical cycling of potassium, phosphorus,

and other soil elements, and is widely used in agriculture, bioleaching, and wastewater treatment.

P. mucilaginosus is able to degrade insoluble soil minerals with the release of nutritional ions and fix nitrogen,

and thus it has been successfully used as a biofertilizer since the 1990s.

The exocellular polysaccharides produced by P. mucilaginosus is also an effective bioflocculant, and thus plays a

potential role in the treatment of wastewater and biohydrometallurgy. (NCBI BioProject: bp_list[1])

(http://bacmap.wishartlab.com/organisms/1395)

WEB REFERENCES:

1. http://jb.asm.org/content/194/10/2777.abstract

2. books.google.co.in/books?isbn=1464964645

http://www.google.co.in/url?sa=t&rct=j&q=&esrc=s&source=web&cd=2&ved=0CDwQFjAB&url=http%3A%2F%2Fwww.paper.edu.cn%2Fen_releasepaper%2FdownPaper%2F201012-617.html&ei=IugVUZHPCYiurAea4YGYAQ&usg=AFQjCNF3Ss2x6gRMUWo84TFH0uwdUnfVPw&bvm=bv.42080656,d.bmk




http://bacmap.wishartlab.com/organisms/1395

http://jb.asm.org/content/194/10/2777.abstract

3. lib.bioinfo.pl/meid:414420/pmid

4. www.b-paper.com/tag/paenibacillus-mucilaginosus

5. www.bacterio.cict.fr/p/paenibacillus.html

6. libra.msra.cn/Detail?entitytype=1&searchtype=5&id=41919359

7. ijs.sgmjournals.org/content/55/6/2351.abstract

8. connection.ebscohost.com/c/reference-entries/62157871/primer

9. libra.msra.cn/Detail?entitytype=1&searchtype=5&id=39795452

10. Thiobacillus ferrooxidans

Common name

NCIM 5068 Thiobacillus ferrooxidansTemple and Colmer

Synonynms:

"Ferrobacillus ferrooxidans" Leathen and Braley 1954

"Ferrobacillus sulfooxidans" Kinsel 1960

Acidithiobacillus ferrooxidans (Temple and Colmer 1951) Kelly and Wood 2000

Thiobacillus ferrooxidans Temple and Colmer 1951 (Approved Lists 1980)

Tiobacillus ferroxidant

Ferrobacillus sulfooxidans Kinsel Be

Nathanson (1902) was the first microbiologist to isolate a member of the bacterial genus Thiobacillus, a bacteria noted

for its ability to oxidize sulfur. It was not until the late 1940s however that Thiobacillus ferrooxidans was isolated by

Colmer and Hinkle (1947). It was subsequently found to oxidize both sulfur and iron. The bacteria has been

characterized by Colmer and Hinkle (1947), Colmer et al. (1950) and Temple and Colmer (1951) found the bacteria to be

gram-negative, acidophilic and rod-shaped.

Chemical name (IUPAC)

NA

Structural formula

NA

Molecular formula

NA

Molecular mass

NA

Appearance, color, odor, physical state

T. ferrooxidans is an autotrophic, acidophilic, obligately aerobic mesophile occurring in single or occasionally in pairs. It

is highly motile with a single flagellum & pili, is non sporing and has a genome of about 2.8 X 106 base pairs and 55-

65% of G-C content. T. ferrooxidans grown in 9K medium (with 9PPM of Fe) and derives its biosynthetic requirements

of carbon from atmospheric carbon dioxide and energy by the oxidation of reduced valence inorganic sulfur compounds

or ferrous ions.

.

Boiling point, melting point, vapor pressure, density

NA

Solubility in water and organic solvents

NA

Viscosity (liquid form)

NA

Hydrolysis

It is susceptible to both biodegradation and hydrolysis

Photolysis

http://www.b-paper.com/tag/paenibacillus-mucilaginosus

http://www.bacterio.cict.fr/p/paenibacillus.html

Photolysis is part of the light-dependent reactions of photosynthesis.

Under normal sunlight Thiobacillus ferrooxidans does not change.

Half life (DT50)

Thiobacillus ferrooxidans (AgriLife Fe Sol B) is not a chemical pesticide.

Appearance, color, odor, state

T. ferrooxidans is an autotrophic, acidophilic, obligately aerobic mesophile occurring in single or occasionally in pairs. It

is highly motile with a single flagellum & pili, is non sporing and has a genome of about 2.8 X 106 base pairs and 55-

65% of G-C content. T. ferrooxidans grown in 9K medium (with 9PPM of Fe) and derives its biosynthetic requirements

of carbon from atmospheric carbon dioxide and energy by the oxidation of reduced valence inorganic sulfur compounds

or ferrous ions.

Solubility in water and organic solvents

NA

Density, Viscosity (liquid form)

NA

Specification

1x 10^ 12 CFU/g

Production process (summarized)

Maintenance of the culture

The efficient Iron solubilizing Thiobacillus ferrooxidans strains used for the mass multiplication were maintained in the

microbiology laboratory as slant cultures and as glycerol stocks and supplied to the production department whenever

required.

Mass multiplication of the biofertilisers

After getting the suitable strains the production process was fine tuned

The culture of T. ferrooxidans used was originally obtained from NCIM,Pune.

To eliminate the possibility of having a mixed or contaminated culture (Guay & Silver, 1975; Tsuchiya, Trivedi &

Schuler, 1974), the strain used in this work was purified by a modification of the slide chamber technique (Postgate,

Crumpton & Hunter, 1961) which allowed the development of a bacterial colony from a single organism to be followed

microscopically. A 5 cm Petri dish containing 2.5 ml solid medium was placed in a metal holder which could be used on

an ordinary microscope stage. Lugs on the holder were aligned with reference marks on the plate to ensure that the plate

could be relocated precisely if movement occurred during incubation. The plates were inoculated with a dilute

suspension of T. ferrooxidans and the positions of single organisms were identified by vernier scale readings. The plates,

in their holders, were put inside larger Petri dishes containing damp filter paper to prevent desiccation of the medium.

These were placed in a glass anaerobic jar under air/N,/CO, (1 :94: 5, by vol.) and incubated at 300C for

36 h. Colony growth of T.ferrooxidans was favoured by conditions of reduced oxygen tension (Mackintosh,

unpublished observation). After incubation the plates were observed microscopically and the positions of

microcolonies were noted. The plates were then re-incubated under the same conditions for 7 d during which time the

microcolonies grew into visible colonies. A deep brown precipitate of ferric sulphate within and around the colony

identified the organism as T. ferrooxidans. Organisms from one of these colonies were picked and inoculated into a flask

of liquid medium and the culture was incubated at 300C for 5 d. The whole procedure was repeated and the final strain

was considered to be uncontaminated T. ferrooxidans.

Media. For the growth of the inoculum, a 'low phosphate' salts medium was used containing: (NH4)S04,

1.0 mM; KH2P04, 0 .2 mM; MgCl2, 125 µM; CaCl2, 1.0 mM; MnCI2, 0.5 µM; ZnCl2, 0.5 µM; CoCI2, 0.5 µM;

H3BO3, 0.5 µM; Na2MoO4, 0.05 µM; CuCI2, 0.5 µM; H2S04,9 .6 mM; FeSO4, 180 mM; pH 1.8.

This medium considerably reduced the amount of precipitate of basic iron sulphates formed during growth and also

decreased the loss of bacteria which adsorb to this precipitate.

Except where otherwise specified, shaken flasks were shaken at 240 rev/min on a rotary shaker describing a circle 3 cm

in diameter. Titratable acidity, expressed as normality of acid present, was followed

as an index of growth of the culture.

Radioactivity of samples dried in aluminum planchets was measured with a gas-flow scaler (Compumatic V, Tracerlab

Inc., Waltham, Mass.).

http://reaction/

http://photosynthesis/

When a good growth was observed the cells were harvested and mixed with the carrier material.

Preparation of the carrier material:

Good carrier material should possess the following criteria:

• Locally available

• High organic matter content with good moisture retaining capacity (50 %)

• No toxic substance

• Easy to process and friable

The cured material was packed in polythene covers and sealed using an electric sealer.

The polythene bags were marked with the name of the product, name of the manufacturer, strain number, recommended

crops, method of inoculation, date of manufacture, expiry date, price and full address of the manufacturer.

ANALYTICAL METHOD

MATERIALS AND METHODS The microbes present in the Sample (Iron solubilizing isolates) (FeSB) are tested on different insoluble Iron compounds.

The solubilization potential was assessed both qualitatively and quantitatively under in vitro conditions.

Plate assay The samples were inoculated into agar medium containing 0.1% insoluble Iron compounds viz FeSO4, FeCl3, and Fe3O4

and incubated at 30ºC for 48 hours. The diameters of the clearing zones around the colonies were measured.

Broth assay The bacterial isolate was inoculated separately to basal medium supplemented with 0.1% insoluble Iron compounds. The

solubilization of Iron from laboratory grade FeSO4, FeCl3, and Fe3O4 by FeSB was assessed. Basal medium was

prepared, splitted in 50 ml aliquots in 100 ml Erlenmeyer flasks and 0.1% of these chemicals were added, steam

sterilized for 30 minutes in an autoclave for 3 consecutive days. Then the flasks were inoculated with 1 ml suspension of

the test culture with a cell load of 107cells ml

-1. Three flasks were maintained with an un-inoculated control for each

treatment. Experiments were done in triplicate. The samples were withdrawn at 0, 7, 14 and 20 days intervals,

centrifuged to remove the debris and cells. Ten ml of this solution was fed to Atomic Absorption Spectro-photometry

(AAS) for determination of the available Iron content.

Determination of pH The pH of the FeSB culture filtrates and the un-inoculated samples was determined at 0, 5, 10 and 15 days after

inoculation. The culture was filtered using Whatman No.1 filter paper. The pH was estimated using Elico pH meter.

Identifying Characteristics

Scanning Electron Microscopy

Strictly aerobic.

Gram-negative.

Polarly flagellated.

Rod shaped.

Motile.

- to 3-

Non-spore forming.

Best growth at 25-

Able to oxidize sulfide, elemental S, thiosulfate, and polythionite.

Obligate autotrophs (cannot grow with organic carbon as an electron and carbon source).

A few species grow on organic compounds.

Grow best at neutral pH.

Some spp. are able to live in highly acidic environments.

Responsible for acid mine drainage due to the metabolism of coal spoil piles causing the production of sulfuric

acid.

Some spp. are able to utilize iron as an energy source.

Some spp. are able to carry out denitrification.

Taxonomic Description

The genus Thiobacillus can be broken into two groups. The first of these are those that grow only at neutral pH values.

These are responsible for the oxidation of elemental sulfur in the reaction:

S- + 202 + 2H2O = 2H2SO4

This process yields 236 kcal of energy. The reduced sulfur compounds are complexed with a sulfhydryl group on a

tripeptide glutathione. It is then oxidized to sulfite with the help of the enzyme sulfide oxidase. There is an apparent

disparity in the lifestyles of these organisms. It lies in the fact that they produce sulfuric acid, but find it toxic. Some of

those organisms growing at lower pH values can however, utilize Fe2+

as an electron donor.

The five most described members of the Thiobacillus species are: Thiobacillus thioparus, Thiobacillus denitrificans,

Thiobacillus thiooxidans, Thiobacillus intermedius, and Thiobacillus ferrooxidans.

Thiobacillus thioparus exhibits the following reaction:

N-C-S- + 2O2 + 2H2O = SO4

2+ NH4

+ + CO2 + 220 kcal

Thiobacillus denitrificans differs in that it can utilize NO3 instead of O2. This denitrification is shown in the following

chemical equation:

2NO3- + S + H2O + CaCO3 = CaSO4 + N2

Thiobacillus thiooxidans has a much more acidic growth range. It grows beat between 2 to 5. It is also strictly aerobic,

and is motile.

Thiobacillus intermedius is a facultative chemolithotroph with a pH range of 3 to 7. It growth is powered by S2O32-

,

which acts as an electron donor, and it is stimulated by the presence of organic matter.

Thiobacillus ferrooxidans is a unique organism when taken in the paradigm of Thiobacillus. It is strictly aerobic and it

has a pH growth range of 1.5 to 5. It can oxidize Fe2+

.

T. Ferrooxidans growth conditions (Horan, 1998)

ENVIRONMENTAL

CONDITIONS Range/Requirement

pH 1-5

Temperature 10 to 37 °C

Energy Source Reduced Sulfur and Ferric Ion

Oxygen Obligate aerobe

Nitrogen source Ammonium salts, nitrate

Almost all of the isolated strains of Thiobacillus ferrooxidans, but none of the T. thiooxidans strains,are able to utilize

molecular nitrogen as a nitrogen source.

The following limits of heavy metal tolerance of T. ferrooxidans were observed:

Cu

Zn

Ni

U

0.87 mol/l

1.83 mol/l

0.85 mol/l

0.004 mol/l

0.05 mol/l

55 g/l

120 g/l

l50 g/l

1 g/l without adaptation

12 g/l after adaptation

Mo 0.0008 mol/l 0.08 g/l

Bio Chemical Tests:

TEST RESULT

Gram Stain Gram Negative

MacConkey +ve

Oxidase +ve

Catalase +ve

Starch Hydrolysis -ve

Methyl Red -ve

Voges-Proskauer +ve

Simmons Citrate +ve

Oxidation-Fermentation +ve/+ve

Triple Sugar Ion Acid/Acid

No gas/No H2S

Indole -ve

Gelatin +ve

Urease -ve

Nitrate +ve

Motility -ve

REVIEW OF LITERATURE:

The beneficial effects of application of apatite along with sulphur and its oxidizing bacteria (Thiobacillus) to enhance

nutrient availability (P, Fe, Zn,...) and in turn uptake of these nutrients by plants has been showed repeatedly by many

researchers (Pathirathna et al., 1989; Schofield et al., 1981; Bardiya et al., 1982; Swaby, 1975

How T. ferroxidans works ?

T ferroxidans is applied and upon exposure to the air, pyrite undergoes oxidation as follows:

(1) FeS2(s) + (7/2)O2 + H2O = Fe2+ + 2SO42- + 2H+

The ferrous iron is further oxidized to ferric iron. This is precipitated if the environmental pH is higher than about 3.

(2) Fe2+ + (1/4)O2 + H+ = Fe3+ + (1/2)H2O

(3) Fe3+ + 3H2O = Fe(OH)3(s) + 3H+

Thus, the overall reaction is:

(4) FeS2(s) + (15/4)O2 + (7/2)H2O = Fe(OH)3(s) + 2SO42- + 4H+

producing four equivalents of acidity from the oxidation of one mole of pyrite.

It is known that the reaction (2) is a very slow process if it proceeds in a purely chemical fashion. This is the reason why

T ferroxidans is needed to be applied to Aqua pond to hasten the process of oxidation.

In the absense of treatment with T ferroxidans, at a pH of 3, the half life of this reaction is around 1000 days (Stumm and

Morgan 1970).,

However, this reaction is hastened by autotrophic iron bacteria, Thiobacillus ferrooxidans which is explained by the

following reaction:

(5) FeS2 + 14Fe3+ + 8H2O = 15Fe2+ + 16H+ + 2SO42-

This produces even more acidity. The reaction runs quite rapidly. Due to treatment with T ferroxidans, the half life of this

reaction is reduced to 20 to 1000 minutes (from 1000 days). The oxidation of sulfur in this reaction is thus facilitated by

autotrophic bacterium, Thiobacillus thiooxidans.

Stumm and Morgan (1970) provided the following schematic drawing of the overall process of pyrite oxidation

They stated that:

"To initiate the sequence, pyrite is oxidized directly by oxygen (a) or is dissolved and then oxidized (a'). The ferrous iron

formed is oxygenated extremely slowly (b) and the resultant ferric iron is rapidly reduced by pyrite (c), releasing

additional acidity and new Fe (2) to enter the cycle via (b). Once this sequence has been started, oxygen is involved only

indirectly in the reoxidation of ferrous iron (b), the oxygenation of FeS2 (a) being no longer of significance. Precipitated

ferric hydroxide serves as a reservoir for soluble Fe (2) (d). If the regeneration of Fe (2) decreases, it will be replenished

by dissolution of solid Fe(OH)3".

The reaction (b) is mediated by iron bacteria T ferrooxidans , and thus it may not be seriously rate limiting. But it has to

be noted that to make smooth pyrite oxidation possible, it is necessary to keep the ferric iron activity high by treatment

with T ferroxidans. In this relation, Murakami (1965) remarked that it is noteworthy that liming at an early stage can

retard the oxidation of pyrite.

This microbe contains a rus operon.

This operon consists of genes for

A periplasmic cytochrome (formerly known as cyt c-552 of the family of diheme cytochromes C4)

A high molecular weight outer membrane located cytochrome bc1,

A low molecular weight blue copper-containing protein Rusticyanin (RCy)

An aa3-type cytochrome oxidase,

A high potential iron sulphur protein (HiPIP)

Expression of the rus operon is 5- to 25-fold higher during growth on iron compared with sulfur.

About 5% of the total cell protein of the microbe present in Fe Sol B when grown on iron consists of Rusticyanin.

Rusticyanin may function as an electron reservoir in such a way that it readily takes up electrons available at the outer

membrane and channels them down the respiratory pathway. It serves as redox buffering function and ensures that the

outer membrane Cyc2 electron acceptor remains in a fully oxidized state, ready to receive electrons from ferrous iron.

This may happen even in the presence of short-term fluctuations of oxygen. Iro gene for a high redox potential iron

oxidase (HiPIP) may also play a role.

Iron disulphide is converted to ferric iron mainly by Thiobacillus ferrooxidans.

The conjoint use of FeSO4, ZnSO4, FYM and Thiobacillus ferrooxidans significantly increased the DTPA-Zn and

DTPA-Fe in the saline-sodic soil. The application of micronutrient fertilizers had pronounced effect on release of

ammoniacal, nitrate nitrogen and available phosphorus in the soil.

(Jagtap P.B. Patil J.D. Nimbalkar, C.A., Kadlag A.D.; Effect of micronutrient fertilizers on release of nutrients in saline-

sorlic soil; Journal of the Indian Society of Soil Science

Year : 2006, Volume : 54, Issue : 4)

Are they Aerobic or Anaerobic ?

They are strictly aerobic

Are they Gram negative or Gram Positive ?

They Are Gram-negative.

How Are they Shaped ?

They are Rod shaped.

What is the normal size of the cell ?

Cells are 0.3 - 3 microns in size.

Do they form spores ?

They are Non-spore forming type

What is the ambient temperature ?

Can achieve best growth at 25-35 degree C

What is the ambient pH ?

Grow best at a pH of 1.5 to 5

What is the way to monitor Fe++

Analyse Fe ++ in the soil/ pond before treatment. Treat. Measure Fe++ in the soil/ pond once in 2 Days till it is

controlled

Do's and Don't's while using T. Ferroxidans

Do not use any sanitisers, disinfectants etc while using T ferrooxidans.

Compatibility of T Ferroxidans with other microbes

Compatible with most of the beneficial microbes.

Stability of the formulation

Stable for about Six months from date of mfg at around 20 Deg C.

Cultures of Thiobacillus ferrooxidans and Thiobacillus thiooxidans, are very difficult to preserve by conventional

methods. Hence, to preserve the cultures with their activity intact, various techniques were tried, after determining their

respective activity in terms of Iron Oxidation Rate (IOR) and Sulfur Oxidation Rate (SOR). Among the methods tested,

along with the recommended method of serial transfer in a liquid medium, were methods such as lyophilization, storage

in a liquid nitrogen and mixing with sterile, inert carriers like lignite or chalcopyrite ores. After a period check-up at 4

months and 8 months storage, it was found that out of these methods, mixing with sterile ore followed by storage at 8

degrees C, kept both types of activities intact. The temperature of storage was observed to have a definite effect on

activity, in that when the preserved cultures were stored at 8 degrees C, the activity was retained, whereas at 28-30

degrees C (RT) storage, the activity of all the cultures preserved by various techniques, dropped significantly

Thiobacillus ferrooxidans bacteria, which grow on a wide spectrum of substrates including the reduced forms of metals

(Fe2+, Sn2+, Sb3+, U4+) or reduced forms of inorganic sulphur (S°, S2-, S2O3 2-), permit to select the mutants of

specific biochemical properties. They have become an attractive material of possible applications in different industrial

processes.

DESULPHURICATION

Recently, it has been found that the metabolism of Thiobacillus ferrooxidans can be used in the technology of

desulphurication of fuels and industrial gasses.

The efficiency of this process is competitive with the traditional methods of desulphurization and the side products are

sulphur, sulphuric acid or gypsum.

Oxidation of elemental Sulfur by Thiobacilli

Coal often contains considerable amounts of pyrite which on combustion is oxidized to sulfur dioxide. To minimize the

emission of this toxic and acid forming gas Dr. Ebner and his co-workers in a laboratory of the Bergbau-Forschung in

Essen try to desulfurize coal by bacterial leaching.

LEACHING IRON

Thiobacillus ferrooxidans is found to be an effective microbe that releases certain Factors to break Ferrous Sulphide to

Ferrous Oxide

T ferroxidans is applied and upon exposure to the air, pyrite undergoes oxidation as follows:

(1) FeS2(s) + (7/2)O2 + H2O = Fe2+ + 2SO42- + 2H+

The ferrous iron is further oxidized to ferric iron. This is precipitated if the environmental pH is higher than about 3.

(2) Fe2+ + (1/4)O2 + H+ = Fe3+ + (1/2)H2O

(3) Fe3+ + 3H2O = Fe(OH)3(s) + 3H+

Thus, the overall reaction is:

(4) FeS2(s) + (15/4)O2 + (7/2)H2O = Fe(OH)3(s) + 2SO42- + 4H+

producing four equivalents of acidity from the oxidation of one mole of pyrite.

It is known that the reaction (2) is a very slow process if it proceeds in a purely chemical fashion. This is the reason why

T ferroxidans is needed to be applied to hasten the process of oxidation.

In the absense of treatment with T ferroxidans, at a pH of 3, the half life of this reaction is around 1000 days (Stumm and

Morgan 1970).,

However, this reaction is hastened by autotrophic iron bacteria, Thiobacillus ferrooxidans which is explained by the

following reaction:

(5) FeS2 + 14Fe3+ + 8H2O = 15Fe2+ + 16H+ + 2SO42-

This produces even more acidity. The reaction runs quite rapidly. Due to treatment with T ferroxidans, the half life of this

reaction is reduced to 20 to 1000 minutes (from 1000 days). The oxidation of sulfur in this reaction is thus facilitated by

autotrophic bacterium, Thiobacillus thiooxidans.

Stumm and Morgan (1970) provided the following schematic drawing of the overall process of pyrite oxidation

They stated that:

"To initiate the sequence, pyrite is oxidized directly by oxygen (a) or is dissolved and then oxidized (a'). The ferrous iron

formed is oxygenated extremely slowly (b) and the resultant ferric iron is rapidly reduced by pyrite (c), releasing

additional acidity and new Fe (2) to enter the cycle via (b). Once this sequence has been started, oxygen is involved only

indirectly in the reoxidation of ferrous iron (b), the oxygenation of FeS2 (a) being no longer of significance. Precipitated

ferric hydroxide serves as a reservoir for soluble Fe (2) (d). If the regeneration of Fe (2) decreases, it will be replenished

by dissolution of solid Fe(OH)3".

The reaction (b) is mediated by iron bacteria T ferrooxidans , and thus it may not be seriously rate limiting. But it has to

be noted that to make smooth pyrite oxidation possible, it is necessary to keep the ferric iron activity high by treatment

with T ferroxidans. In this relation, Murakami (1965) remarked that it is noteworthy that liming at an early stage can

retard the oxidation of pyrite.

LEACHING TIN

Laboratory investigations confirm that it is possible to leach tin from synthetic minerals like stannite, kesterite,

stannoidite, herzenbergite, ottemannite and berndtite, and from natural tin minerals which include stannite, cassiterite and

varlamoffite, in the presence ofThiobacillus ferrooxidans, and with organic agents of biological origin, especially oxalic

acid and oxalic-citric acids mixture.

Over a leaching period of 35 days with 0.5% pulp density, initial pH of 2.5, using minus 0.16 mm size fraction at 32°C,

as much as 54.45, 72.66, 97.13, and 31.30% Sn were extracted from synthetic stannite, kesterite, stannoidite and natural

stannite, respectively. Varlamoffite, found in the dried, leached residues of the tin sulphides, provides evidence that

bacterial action can be responsible for the genesis of supergene varlamoffite.

LEACHING URANIUM:

Uranium(IV) for example as uranium dioxide UO2, uraninite, is oxidized by ferric iron to uranium(VI) and so soluble

uranyl ions UO2 are formed:

UO2 + 2Fe+++

à (UO2)++

+ 2 Fe++

LEACHING COPPER

In general, the leaching of metals from chalcopyrite occurs according to the reactions given below.

It has been established that leaching of copper and iron from chalcopyrite takes place by both direct enzymatic oxidation

of iron and sulphur (reaction 1 and 2) and indirect

chemical dissolution of copper according to the reaction 3.

The Thiobacillus ferrooxidans bacteria live on the energy from the process of oxidation of Fe (II) to Fe (III) and the

sulphide sulphur to sulphate sulphur. In this way the

cycle of transformation of Fe (II) to Fe (III) is closed, and the formed H2SO4 increases the solubility of the mineral.

The chemical leaching of iron occurs with a yield from 8% (1% mineral in the medium) to 5% (1.7% mineral in the

medium), while the chemical leaching of copper yields about 2.5%, irrespective of the concentration of chalcopyrite in

the mixture

The following limits of heavy metal tolerance of T. ferrooxidans were observed:

Cu

Zn

Ni

U

Mo

0.87 mol/l

1.83 mol/l

0.85 mol/l

0.004 mol/l

0.05 mol/l

0.0008 mol/l

55 g/l

120 g/l

l50 g/l

1 g/l without adaptation

12 g/l after adaptation

0.08 g/l

Toxicology

Toxicity class WHO

Not Listed in PAN Bad Actors. (PAN Bad Actors are chemicals that are one or more of the following: highly acutely

toxic, cholinesterase inhibitor, known/probable carcinogen, known groundwater pollutant or known reproductive or

developmental toxicant. NOTE! Because there are no authoritative lists of Endocrine Disrupting (ED) chemicals, EDs

are not yet considered PAN Bad Actor chemicals.)

LD50 acute oral

NA

LD50 acute dermal

NA

LC50 acute inhalation

NA

Eye irritation

Non-irritating

Skin irritation

Non-irritating

Sensitization

Not a skin sensitizer

Carcinogenicity

It is not carcinogenic

Ingredients not listed by ACGIH, IARC, NIOSH, NTR, or OSHA

Mutagenicity

NA

Teratogenicity

NA

Effect on reproduction

NA

I.3. RESIDUE AND EFFECT ON HUMAN According to human health, NCIM 5068 Thiobacillus ferrooxidans is considered non-pathogenic.

Cholinesterase Inhibitors: No

Metabolise in animal and human body

Not Available

Metabolise and degradation in plant, soil

Not Available

Residue data from other countries

Not Available

Residue analytical method on crops

Not Available

Fat tissue accumulate

Not Available

Max Residue Limit (MRLs)

Not Available

Acceptable daily intake (ADI)

http://www.pesticideinfo.org/Docs/ref_toxicity7.html#BadActor

Not Available

Pre-harvest interval (PHI) (into finished product)

Not Available

EFFECT ON ENVIRONMENT

Environment fate

Thiobacillus ferrooxidansis usually inoculated directly into the soil. There, once the bacteria are released from the

inoculant carrier, they are subjected to all the chemical and physical soil factors in addition to competition from the

indigenous soil microflora.

Volatility

Owing to it’s low volatility, evaporation from products containing it will be minimal.

Absorption in the soil

It has minimal tendency to bind to soil or sediment

Leaching GUS leaching potential index

It improves soil fertility. It increases, soil organic matter and water holding capacity.

Soil degradation

NA

Hydrolysis


Photolysis


Under normal sunlight NCIM 5068 Thiobacillus ferrooxidans does not change.

EFFECT ON NON-TAGET ORGANISMS

The biodegradability insures that there is no environmental pollution

Effect on bird, bee, wild animals

It is practically nontoxic to very slightly toxic

Effect on fish, aquatics

Very slightly to moderately toxic

Effect on natural enemies

NOT AVAILABLE

11 Silica solublizing bacteria

Bacillus globisporus Larkin and Stokes

Bacillus globisporus Larkin and Stokes, 1967

Synonyms:

Sporosarcina Globispora

CCM 2119, ATCC 23301, IFO 15682, LMG 6928, DSM 4, JCM 2509, NCIMB 11434, CIP 103.266; CCUG 7419;

NRRL NRS-1533; Stokes W25

http://reaction/


WHAT ARE SILICATES?

Sodium Silicate Calcium Silicate Aluminum Silicate Sandy Soil

A silicate is a compound containing an ion in which one or more central silicon atoms are surrounded by electronegative

ligands. This definition is broad enough to include species such as hexafluorosilicate ("fluorosilicate"), [SiF6]2−, but the

silicate species that are encountered most often consist of silicon with oxygen as the ligand. Silicate anions, with a

negative net electrical charge, must have that charge balanced by other cations to make an electrically neutral compound.

Silica, or silicon dioxide, SiO2, is sometimes considered a silicate, although it is the special case

with no negative charge and no need for counter-ions. Silica is found in nature as the mineral quartz, and its polymorphs.

Silica as Plant Nutrient:

Silicates make the cell walls of the plants thicker and stronger while also increasing the size of the vascular system of the

plant. The thicker cell walls translate to the plant being stronger in all aspects, while the enlarged vascular system can

take up more water and nutrients resulting in a bigger, healthier, higher yielding plant! The larger the plant's vascular

system, the more potential the plant has for maximum yield.

Mostly silica is deposited on leaves and stems of plants, and some effects are through interaction between silicic acid and

other elements such as Al. In contrast to essential elements, the function of Silica in plants is probably mechanical rather

than physiological. This characteristic of Silica function explains why Si effects are easily observed in plants that

accumulate Silica to a certain extent and why Si effects are more obvious under biotic or abiotic stress. With the changes

occurring in the global environment, the role of Silica will become more and more important for better and sustainable

production of crop.

Targets of a suitable Silica fertilizer:

Cheaper Source

Easy application,

Higher content of soluble Silica

Ready availability,

Silica is the second most abundant element in the earth's crust.

It is always combined with other elements and many of these sources are insoluble. Responses of crops to soluble Silica

applications in sands (largely SiO2) provide an example of the insolubility of the sandy soils.

Although basic slags (by-products) from the processing of iron and alloy industries, have been used as source of Si, their

concentrations and solubility of Silica and the contents of other elements and their bondage vary widely.

Potassium silicate is used in nutriculture for disease control in some high value crops but are too costly for general use.

Sodium silicate and silica gel have also been used to supply Silica in research and high value crops.

Calcium silicates have emerged as the most important sources for soil applications. Of those, calcium meta-silicate

(wollasonite, CaSi03) has been the most effective source in many locations with low concentrations of soluble Silica in

soils. Such a material, supplied as a slag by-product from the high temperature electric furnace production of elemental P

is applied extensively to organic and sandy soils for application for sugarcane and rice crops as well as utilization on turf.

Role of silicon in mitigation of / alleviating the abiotic stress in plants

Abiotic stress

Plant/crop

Reference

Physical stress:

Lodging

Drought

Radiation

High temperature

Freezing

UV etc

All crops

Marschner et al., 1990

Chemical stress:

Salinity

Mn toxicity

Al toxicity

Fe toxicity

Rice

Wheat

Mesquite

Bean

Rice

Leaf freckle in sugarcane

Natoh et al., 1986

Ahmad et al., 1992

Bradbury and Ahmad, 1990

Horst and Marschner,1978

Horiguchi,1988

Li et al., 1989

Fox et al.,1967

Diseases suppressed by Si nutrition

Sl.No.

Disease

Pathogen

Reference

Leaf and neck blast

Pyricularia oryzae

Winslow (1992)

Brown spot

Bipolaris oryzae

Dantnoff et

al(1992)

Rice Sheat blight Rhizoctonia solani Dantnoff et

al(1992)

Leaf scald

Gerlachia oryzae

Winslow (1992)

Grain discoloration

Bipolaris fusarium

Winslow (1992)

Stem rot

Sclerotium oryzae Elawad and

Green (1979)

Sugarcane

Sugar rust

Puccinia melanocephala

Dean and Todd,

1979

Ring spot

Leptosphaeria Sacchari

Phyllosticta sp. [anamorph]

Raid et al., 1991

Banana

Panama wilt

Fusarium,oxysporium,f.sp.cubense

Cucumber

Powdery mild dew

Sphaerotheca fuliginea,Pythium.

Adatia and

Besford,1986

Wheat

Powdery mildew

Erysiphe graminis, Oidium

monilioides

Cowpea

rust

Uromyces phaeseoli typia Arth.

Pest suppressed by Si nutrition

Sl.no.

Pest

Scientific name

Reference

Rice

Stem maggot Chlorops oryzae Sawant et al (1994)

Green leaf hopper Nephotettix bipunctatus Maxwell et al (1972)

Brown plant hopper Nilaparvata lugens Sujatha et al (1987)

White backed plant hopper Sogetella furcitera Salim and Saxena

(1992)

Leaf spider Tetranychus spp. Yoshida (1975)

Wheat

Green bug

Scizaphis graminum

Gomes et al.,2005

Sorghum

Green bug

Scizaphis graminum

Carvalho et al.,1999

Corn

Leaf aphid

Rhopalosiphum maidis

Goussain,2001

Sugarcane

Stalk borer

Eldana saccharina

Kvedaras et al., 2005

Role of Microbes:

Bacteria have also been shown to accelerate the dissolution of silicates by the production of excess proton and organic

ligands, but also in some cases by the production of hydroxyl, extracellular polysaccharides (EPS), and enzymes (Welch

et al. 1999, Berthelin & Belgy 1979, Malinovskaya et al. 1990).

http://en.wikipedia.org/wiki/Phyllosticta

Microbially produced organic ligands include metabolic byproducts, extracellular enzymes, chelates, and both simple

and complex organic acids. These substances can enhance silicate dissolution rates by decreasing pH, forming

framework destabilizing

surface complexes, or by complexing metals in solution (Bennett & Casey 1994, Blake &

Walter 1996, Drever & Vance 1994, Stillings et al. 1996). Welch & Ullman (1993) found that the rates of plagioclase

dissolution in solutions containing organic acids were up to ten times greater than rates in solutions containing inorganic

acids at the same acidity.

Microorganisms are capable of utilizing silicate-bound nutrients, and respond to the addition of metal chelating ligands,

which increase silicate weathering and nutrient release.

(http://people.ku.edu/~jenrob/4/files/WRI01.pdf)

MODE OF ACTION:

During the metabolism of microbes that are present in Si Sol B, several organic acids are produced and these have a dual

role in silicate weathering. They supply H+ ions to the medium and promote hydrolysis and the organic acids like citric,

oxalic acid, Keto acids and hydroxy carbolic acids which form complexes with cations, promote their removal and

retention in the medium in a dissolved state.

In the bacterial metabolic pathways, glucose is converted into gluconic acid which is expelled to the environment. The

organic acid greatly enhances solubility of several soil minerals, in particular apatites, dissolution of which releases

phosphate ions to the environment making them available to plants.

2-ketogluconic acid seemed to be the most active agent for the solubilization of the three silicate minerals. Gluconic and

acetic acids were likely involved in the solubilization of feldspar.

The presence of gluconic acid enhances the solubility of pyromorphite. Both acidification and complexing capabilities of

the metabolite play the role in the process. The mineral dissolves more with temperature.

However particle size and soil type have significant effects on the removal of Potassium, Calcium, Aluminum,

phosphorus and Sodium from the Soil.

Biohazard Level:

1

Morphology of Sporosarcina Globispora

Gram positive rods, 0.8-1.0 / 1.3-5.0 μm, motility variable.

Spherical, sub terminal or terminal spore, swelling the sporangium

Growth conditions of Sporosarcina Globispora

Growth temperature from 0-3 ºC to 25-30 ºC.

- no growth in anaerobic agar

- no growth in 5% NaCl

- no growth in 10% NaCl

- no growth at 5.7 pH

Biochemical characters of Sporosarcina Globispora

Acid production from glucose - positive.

Acid production from mannitol, arabinose & xilose - negative

Catalase, deamination of phenylalanine - positive.

Reduction of nitrate to nitrite, casein & starch hydrolysis - variable.

Decomposition of tyrosine, Voges-Proskauer, resistance to lysozyme, citrate utilization &

indole - negative.

TARGET CROPS:

Cereals, grasses, Mango, Paddy, Arrowroot, Avocado, Banana, Pineapple, Squash, Bean, Sunflower, and Cucumber.

SALIENT FEATURES OF Si Sol B

Confers strength, rigidity

Creates better water-use efficiency.

Decreases the transpiration rate (water loss through leaves)

Enhances pollination in tomatoes

Enhances the growth, chlorophyll content, thousand grain weight, filled grains, biomass and yield in rice

Ensures better pollen fertility in cucurbits.

Ensures stronger stems and more erect leaves, which capture more sunlight.

Filters harmful ultraviolet radiation reaching leaf surface

http://people.ku.edu/~jenrob/4/files/WRI01.pdf

Found to alleviate many biotic and abiotic stresses.

Hastens the decomposition of rice straw that is left over after harvesting

Improves plant growth and yield

Improves the oxidizing power of rice roots and accompanying tolerance to high levels of iron and manganese

Increases pest and disease resistance.

Increases rates of photosynthesis

increases root growth in grasses

Increases the grain yield

Increases the photo assimilation of carbon

Increases the photosynthesis by improving light interception.

Increases the synthesis of proteins and chlorophyll.

Increases the uptake of nitrogen (Okomota, 1969; Sadanandan and Verghese, 1969).

Induces resistance to pests and diseases.

Inhibits Sucking pests and leaf eating caterpillars

Plays a role in phosphorus nutrition

Promotes the assimilated carbon to the panicle in rice (Takahashi and Miyake., 1982).

Provides a physical barrier to insects at epidermal layer levels

Reduces chaffiness.

Reduces phosphatase resulting in a greater supply of essential high energy precursors needed for optimum

growth.

Reduces the incidence of fungal attacks even under heavy application of nitrogen in the case of rice plants

Regulates uptake of iron, manganese and aluminum.

Strengthens epidermal cells in leaves and stems.

Suppresses invertase resulting in greater sucrose production in sugarcane

Tends to maintain erectness of rice leaves and clumps t

Recommended Dosage:

Soil Application at Root Zone at the time of land :

Cereals: 2 Kg/ Acre

Horticulture: 25-50g/ tree

Floriculture: 2-3 Kg/ Acre

Sugarcane: 4-5 Kg/ Acre

Oil Seeds: 2-3 Kg/ Acre

Vegetables: 2-3 Kg/ Acre

Foliar Application:

Cereals: 250ml/ Acre

Horticulture: 2-5 ml/ tree

Floriculture: 250-300 ml/ Acre

Sugarcane: 400-500 ml/ Acre

Oil Seeds: 200-300 ml/ Acre

Vegetables: 200-300 ml/ Acre

REFERENCES:

1. Hurlbut, Cornelius S.; Klein, Cornelis (1985). Manual of Mineralogy, Wiley, (20th edition ed.). ISBN ISBN 0-471-

80580-7

2. Gordon R.E., Haynes W.C., Pang C.H. (1973) – The genus Bacillus . Agriculture Handbook No. 427, U.S.D.A.,

Washington D.C.

3. Buchanan R.E., Gibbons N.E., Cowan S.T., Holt J.G., Liston J., Murray R.G.E., Niven C.F., Ravin A.W., Stanier

R.W. ( 1974) – Bergey’s Manual of Determinative Bacteriology, Eight Edition, The Williams & Wilkins Company,

Baltimore.

4. Yoon J.H., Lee K.C., Weiss N., Kho Y.H., Kang K.H. & Park Y.H.: Sporosarcina aquimarina sp. nov., a bacterium

isolated from seawater in Korea, and transfer of Bacillus globisporus (Larkin and Stokes 1967), Bacillus psychrophilus

(Nakamura 1984) and Bacillus pasteurii (Chester 1898) to the genus Sporosarcina as Sporosarcina globispora comb.

nov., Sporosarcina psychrophila comb. nov. and Sporosarcina pasteurii comb. nov., and emended description of the

genus Sporosarcina. Int. J. Syst. Evol. Microbiol., 2001, 51, 1079-1086.

REVIEW OF LITERATURE

http://en.wikipedia.org/wiki/Special:BookSources/0471805807

http://en.wikipedia.org/wiki/Special:BookSources/0471805807

Enhanced resistance to foliar diseases such as ring spot may partially explain yield increases obtained with

application of calcium silicate slag to soils low in plant-available silicon

(R.N. Raid, D.L. Anderson and M.F. Ulloa; Influence of cultivar and amendment of soil with calcium silicate slag on

foliar disease development and yield of sugar-cane; Crop Protection; Volume 11, Issue 1, February 1992, Pages 84-

88)

Silicon nutrition enhanced plant growth and yield. Application of organic siliceous materials like straw, husk,

husk ash enhanced root length, shoot length, plant height, total and productive tillers per hill, filled grains and

1000 grain weight in rice. The increase was found in dry matter and yield. Inoculation of silicate solubilizing

bacteria with these materials recorded further enhancement. Application of potassium, magnesium and calcium

silicate to rice increased the yield. On an average 10-30% increased yields were recorded through silicate

amendments.

The solubilization of silicates was investigated using kaolin and quartz sand as model substances. The mineral

solubilization was studied in the concentration of solubilized Si and Al. The chemical leaching of the silicates

was carried out using inorganic and organic acids as well as sodium hydroxide. The process was more effective

in the alkine than in the acid pH range. In the acid medium, oxalic acid showed maximum acidity and a tendency

to form complex structures, especially with aluminium, and was most effective in leaching.

The microbiological influence on solubilization reactions was tested using a number of microorganisms among them

acid, alkali and slime-forming species. The highest leaching activity was observed in the case of Thiobacillus

thiooxidans, whereas the heterotrophic microorganisms (among them Bacillus mucilaginosus) did not exercise a

sollubilizing effect on the silicates.

X-ray phase analysis of leached kaolin samples did not show any differences from the non-leached mineral.

(S. Friedrich, N. P. Platonova, G. I. Karavaiko, E. Stichel, F. Glombitza; Chemical and microbiological solubilization of

silicates; Acta Biotechnologica; Volume 11, Issue 3 , Pages187 – 196)

A key observation emerging from the data is the presence of significantly higher population of silicate

solubilizing bacteria in the rhizosphere of field-tolerant palms at 0–25 cm depth compared to root (wilt)-diseased

palms. The numbers of these function-specific bacteria were on par at 25–50 cm depth in both types of palms.

This observation is of interest in the sense that it might have implications in nutrition, and thus the physical appearance

of field tolerant vis-à-vis diseased palms. Majority of the silicate solubilizers encountered in our study were Bacillus spp.

and Pseudomonas spp., though at times Aspergillus sp. and Penicillium sp. were also identified. Though the general

bacterial population was high in the root zone of the root (wilt)-diseased palms compared to field-tolerant palms,

function-specific microbes, viz. nitrogen-fixers, phosphate solubilizers, cellulose degraders and silicate solubilizers,

which are mostly bacteria, are present in significantly higher numbers in the rhizosphere of field tolerant palms than in

diseased palms. Careful interpretation revealed that as high as 3.6% of the total bacterial population was made up of

function-specific bacteria in disease-tolerant palms compared to the mere 0.21% in diseased palms. Presence of these

function-specific microbes could be playing a role in improving the nutrient availability to the palms, thereby perhaps

conferring vigour to the field-tolerant palms.

Silica is primarily deposited on the walls of epidermis and vascular tissues and confers strength and rigidity to the leaves,

more particularly so in monocotyledonous coconut crop. In a study at the Central Plantation Crops Research Institute at

Kasaragod, on nutritional management of root (wilt)-affected adult coconut palms with organic manure and silicon

during 1995–98, it was reported that the silicon dioxide content in the leaf samples (14th leaf) of root (wilt)-diseased

palm was consistently found to be around 0.24 to 1.7%, which was significantly lower compared to the leaf of healthy

coconut palm24 containing SiO2 to the tune of 59–134%. In this context, it is important to note that field-tolerant

coconut palms exhibit normal, erect leaves with the leaflets avoiding bending and having abundant chlorophyll content,

while diseased palms exhibit wilting and drooping of leaves, flaccidity, ribbing with yellowing and necrosis of leaflets25.

Though the soils of Kerala, especially the sandy and sandy loams have high silica content, the soil silicate is generally

present in unavailable polymerized forms and for its absorption by plants, it has to be depolymerized and rendered

soluble by means of biological or chemical reactions. A variety of soil microorganisms have been found to solubilize

silicates. These bacteria solubilize the insoluble silicates by production of CO2, organic acids and/or exo

polysaccharides. Gluconate-promoted dissolution of silicates like albite, quartz and koalinite by sub-surface bacteria has

been recorded earlier. Large numbers of silicate-solubilizing bacteria in the root zones of field tolerant palms observed in

this study could improve the available pool of silica (silicic acid, H4SiO4) to be absorbed by these palms, which is

reflected in their firm and erect leaves arranged to receive maximum sunlight. Field-tolerant palms are shown to have

better photosynthetic turnover, perhaps by virtue of higher silica uptake, since silica crystals are implicated in absorbing

optimum photo energy for efficient photosynthetic process in many crops, including monocots. Physiologically, presence

of silica in leaf epidermis provides mechanical strength to the stomata and regulates transpiration rate. Less numbers of

silicate solubilizers in root (wilt)-diseased palms could possibly be one of the reasons for abnormal stomatal activity

permitting higher transpiration in such palms. Absence of sufficient silica cells in the leaves of diseased palms could

cause the stomata to collapse and remain abnormally open.

The vectors of root (wilt) disease, S. typica and P.moesta, introduce the phytoplasma into the palms by inserting the

stylet through the stomatal opening present in the lower epidermis. Here too, the presence of more silica cells in field-

tolerant palms may provide the leaves with higher mechanical and tensile strength, and prevent the vectors from

depositing the pathogen, even though they have been observed to colonize healthy palms. Another physiological

condition is the presence of high levels of free phenols in the roots of healthy palms compared to root (wilt)-diseased

palms. There is evidence to show that more silica in roots promotes more phenol production. Also, presence of silica in

many monocot and vegetable crops provides resistance to fungal diseases. In coconut too, high silica content in field-

tolerant palms might help them evade secondary fungal infection, whereas root (wilt) diseased palms succumb to leaf rot

disease caused by a fungal complex.

The rhizosphere microbial make-up of any plant is dependent upon the rhizodeposition (organic acids and other exudates

produced by the roots), which is again dependent

on the genetics of the plant, its health, soil type and anthropogenic interventions. With the above observations of

differences in the rhizosphere microbial ecology of root

(wilt)-diseased and field-tolerant coconut palms, we would like to hypothesize that the presence of more number of

silicate-solubilizing bacteria in the rhizosphere of fieldtolerant coconut palms could promote more silica uptake, which

may provide tensile strength to the leaves to resist vector feeding and prevent phytoplasma inoculation.

REFERENCES:

1. Solomon, J. J., Govindankutty, M. P. and Neinhaus, F., Association of mycoplasma-like organisms with the coconut

root (wilt) disease in India. Z. Pflkranh. Pflschutz., 1983, 90, 295–299.

2. Anon., Coconut root (wilt) disease. Intensity, production loss and future strategy, CPCRI, Kasaragod, 1985, p. 45.

3. Rajan, P. and Mathen, K., Proutista moesta (Westwood) and other additions to insect fauna on coconut palm. J. Plant.

Crops, 1985, 13, 135–136.

4. Mathen, K., Rajan. P., Nair, C. P. R., Sasikala, M., Gunasekaran, M., Govindankutty, M. P. and Solomon, J. J.,

Transmission of root (wilt) disease to coconut seedlings through Stephanitis typical (Distant) (Heteroptera: Tingidae).

Trop. Agric., 1990, 67, 69–73.

5. Srinivasan, N. and Gunasekaran, M., Incidence of fungal species associated with leaf rot disease of coconut palms in

relation to weather and stage of lesion development. Ann. Appl. Biol., 1996, 129, 433–449.

6. Davis, T. A., Breeding in coconut for disease resistance. Indian Coconut J., 1953, 6, 95–100.

7. Iyer, R. D., Rao, E. V. V. B. and Govindankutty, M. P., Super yielder in coconut. Indian Farm., 1979, 28, 3–5.

8. Anon., Annual Report 1986, Central Plantation Crops Research Institute, Kasaragod, 1988, p. 205.

9. Nair, M. K., Koshy, P. K., Jacob, P. M., Rao, E. V. V. B., Nampoothiri, K. U. K. and Iyer, R. D., A root (wilt)

resistant coconut hybrid and strategy for resistance breeding. Indian Coconut J., 1996, 27, 2–5.

10. Solomon, J. J., Sasikala, M. and Shanta, P., A serological test for the detection of root (wilt) disease of coconut. In

Coconut Research and Development (ed. Nayar, N. M.), Wiley Eastern Ltd, New Delhi, 1983, pp. 401–405.

11. Allen, O. N., In Experiments in Soil Bacteriology, 1959, 3rd edn, p. 117.

12. Martin, J. P., Use of acid, rose bengal and streptomycin in the plate method for estimating soil fungi. Soil Sci., 1950,

69, 215–232.

13. Becking, J. H., Nitrogen fixing bacteria of genus Beijerinckia in South African soils. Plant Soil, 1959, 11, 193–206.

14. Pikovskaya, R. I., Mobilization of phosphorus in soil in connection with vital activity of some soil microbial species.

Mikrobiologiya, 1948, 17, 362–370.

15. Hankin, L. and Anagnostakis, S. L., Solid media containing carboxymethylcellulose to detect CX cellulase activity of

microorganisms. J. Gen. Microbiol., 1977, 98, 109–115.

16. Bunt, J. S. and Rovira, A. D., Microbiological studies of some subantartic soils. J. Soil Sci., 1955, 6, 119–128.

17. Potty, V. P., George, M. and Jayasankar, N. P., Effect of crop mixing on coconut rhizosphere. Indian Coconut J.,

1977, 8, 1–2.

18. Alice, E. J., Karunakaran, P. and Samraj, J., A comparative study of the rhizosphere microflora of coconut palms

from diseased and healthy areas with reference to root (wilt). Indian Coconut J., 1980, 11, 1–4.

19. Gopal, Murali, Gupta, Alka, Nair, C. P. R. and Rajan, P., Effect of systemic soil insecticides and plant product on

microbial load of soil in root (wilt) affected coconut monocropping ecosystem. Coconut Res. Dev., 2001, 17, 52–71.

20. Nagaraj, A. N. and Menon, K. P. V., Observation on root decay in coconuts, its cause and its relation to the foliar

symptoms of disease in the disease belt of Travancore–Cochin. Indian Coconut J., 1955, 8, 97–105.

21. Maramorosch, K., A survey of coconut disease of uncertain etiology. Report FAO, UN, Rome, 1964, p. 39.

22. Joseph, T. and Jayasankar, N. P., Evaluation of root degeneration in

coconut in relation to root (wilt) disease. Plant Dis., 1981, 66,

666–669.

23. Anon., Annual Report 1997, Central Plantation Crops Research Institute, Kasaragod, 1997, p. 45.

24. Verghese, E. J., Sankaranarayanan, M. P. and Menon, K. P. V., Chemical studies on the leaf and root (wilt) disease of

coconut in Travancore–Cochin. II. Nutrient contents of leaves of healthy and diseased palms. In Proc. First. Conf.

Coconut Research Workers,

Trivandrum, 1959, pp. 18–23.

25. Menon, K. P. V. and Pandalai, K. M., In The Coconut Palm – A Monograph, Indian Central Coconut Committee,

Ernakulam, 1958, p. 394.

26. Pillai, T. N. V., Silica content in coconut (Cocos nucifera) kernel and water. Curr. Sci., 1967, 24, 667–668.

27. Jones, L. H. P. and Handrecht, K. A., Silica in soils, plants, and animals. Adv. Agron., 1967, 19, 107–149.

28. Vandevivere, P., Welch, S. A., Ullman, W. J. and Kirchman, D. L., Enhanced dissolution of silicate minerals by

bacteria at nearneutral pH. Microbial Ecol., 1994, 27, 241–251.

29. Dwivedi, R. S., Mathew, C., Michael, K. L., Ray, P. K. and Amma, B. S. K., Carbonic anhydrase, carbon

assimilation and canopy structure in relation to nut yield of coconut. Indian Natl. Sci. Acad. Symp. on Photosynthesis

and Productivity, Lucknow, 1978, pp. 44–46.

30. Epstein, E., Silicon. Annu. Rev. Plant Physiol. Plant Mol. Biol., 1999, 50, 641–664.

31. Rajagopal, V., Patil, K. D. and Amma, B. S. K., Abnormal stomatal opening in coconut palms affected with root

(wilt) disease. J. Exp. Bot., 1986, 37, 1398–1405.

32. Rajagopal, V., Amma, B. S. K. and Patil, K. D., Water relations of coconut palms affected with root (wilt) disease.

New Phytol., 1987, 105, 289–293.

33. Joseph, K. V. and Jayasankar, N. P., Polyphenol content in coconut roots in relation to root (wilt) disease. J. Plant.

Crops, 1973, 1, 99–101.

34. Watteau, F., Villemin, G., Mansot, J. L., Ghanbaja, J. and Tontain F., Localization and characterization by Electron

Energy Loss Spectroscopy (EELS) of the brown cellular substances of beech roots. Soil Biol. Biochem., 1996, 28, 1327–

1332.

35. Belanger, R. R., Bowen, P. A., Ehret, D. L. and Menzies, J. G., Soluble silicon. Its role in crop and disease

management of greenhouse crops. Plant Dis., 1995, 79, 329–336.

(Murali Gopal, Alka Gupta and R. V. Nair; Variations in hosting beneficial plant-associated microorganisms by root

(wilt)-diseased and field-tolerant coconut palms of West Coast Tall variety; CURRENT SCIENCE, VOL. 89, NO. 11, 10

DECEMBER 2005)

Silicate solubilizing bacteria

Microorganisms are capable of degrading silicates and aluminum silicates. During the metabolism of microbes

several organic acids are produced and these have a dual role in silicate weathering. They supply H+ ions to the

medium and promote hydrolysis and the organic acids like citric, oxalic acid, Keto acids and hydroxy carbolic acids

which from complexes with cations, promote their removal and retention in the medium in a dissolved state.

The studies conducted with a Bacillus sp. isolated from the soil of granite crusher yard showed that the bacterium is

capable of dissolving several silicate minerals under in vitro condition. The examination of anthrpogenic materials like

cement, agro inputs like super phosphate and rock phosphate exhibited silicate solubilizing bacteria to a varying degree.

The bacterial isolates made from different locations had varying degree of silicate solubilizing potential. Soil inoculation

studies with selected isolate with red soil, clay soil, sand and hilly soil showed that the organisms multiplied in all types

of soil and released more of silica and the available silica increased in soil and water. Rice responded well to application

of organic sliceous residue like rice straw, rice husk and black ash @ 5 t/ha. Combining SSB with these residues further

resulted in increased plant growth and grain yield. This enhancement is due to increased dissolution of silica and

nutrients from the soil.

(http://agritech.tnau.ac.in/org_farm/orgfarm_biofertilizertechnology.html)

Proteus mirabilis found to take up little amounts of Silicates. In combination with Bacillus caldolyticus it could

take more silicates.

Diatoms can remove 25% of the dissolved Silica from the water medium.

Dissolution of Silicates was achieved by complexation of cationic components of the silicates by 2-

ketagluconate by microorganisms like Erwinia herbicola and Pseudomonas.

Gluconic acid producing bacteria have been shown to solubilize bytownite, albite, kaolinite, quartz at neutral pH.

(Vandevivere et al., 1994)

Fungi and Bacteria which can form Oxalic, Pyruvic and humic acids exhibited slow dissolution of Quartz.

Weathering of spodumene was shown by Arthrobacter pascens, A. globiformis, A. simplex, Nocardia globerulla,

Pseudomonas flourescens, Ps. Putida, Ps. Testorinii, Trichoderma lignorum, Cephalosporum atrum, Pencillium

decumbens.

Pencillium notanum, Aspergillus niger, Bacillus mucilaginosus var. siliceous are the active organisms to degrade

spodamene.

(Karavaiko et al., 1989; Avakyan et al.,1986)

Botritis, Mucor, Pencillium and Trichoderma isolated from Rock surfaces and weathered stone were found to

mobilize silicon of Ca, Mg and Zn Silcates.

(Webley et al., 1963)

Penicillium simplicissimus released Si from basalt, granite, grandiorite, rhyolite, andesite, peridotite, dunite and

quartzite with metabolically produced citric acid

(Silverman and Munoz, 1970)

Pseudomonas mendocina was able to enhance mobilization of Al, Si and Fe impurities from Kaolinite .

(Maurice et al., 2001).

A silicate mineral-solubilizing bacterial strain Q12 was isolated from the surfaces of weathered feldspar and

identified as Bacillus globisporus Q12 based on the 16S rDNA gene sequence analysis. Three silicate minerals

(feldspar, muscovite, and biotite) were used to investigate potassium and silicon mobilization by strain Q12. In

liquid cultures, the strain showed better growth on the biotite than on feldspar and muscovite. The biotite was the

best potassium source for growth of the strain. Solubilization of potassium and silicon from the silicate minerals

by the strain resulted mostly from the action of organic acids. Gluconic acid seemed to be the most active agent

for the solubilization of the 3 silicate minerals. Gluconic and acetic acids were likely involved in the

solubilization of feldspar. The strain could be acid or alkali and salt tolerant and temperature resistant.

(Sheng XF, Zhao F, He LY, Qiu G, Chen L.; Isolation and characterization of silicate mineral-solubilizing Bacillus

globisporus Q12 from the surfaces of weathered feldspar;

Can J Microbiol. 2008 Dec;54(12):1064-8.)

12. Thiobacillus thiooxidans Synonyms:

Acidithiobacillus thiooxidans (Waksman and Joffe 1922) Kelly and Wood 2000, "Thiobacillus crenatus" Emoto 1929,

Thiobacillus concretivorus Parker 1945,

"Thiobacillus lobatus" Emoto 1929,

"Thiobacillus umbonatus" Emoto 1929",

"Thiobacterium thiooxydans" (Waksman and Joffe 1922) Lehmann and Neumann 1927, "Thiobacillus thermitanus"

Emoto 1928,

Thiobacillus thiooxidans Waksman and Joffe 1922

Thiobacillus thiooxidans is an acidophilic, obligately autotrophic bacterium which derives its energy by

oxidizing reduced or partially reduced sulfur compounds and obtains its carbon by fixing carbon dioxide from

the atmosphere.

It is able to live in inorganic, acidic environments and is present in large numbers in coal mine drainage and in

mineral ores.

It is restricted to habitats where both an acceptable electron donor (reduced sulfur compounds or iron in some

cases) and an electron acceptor (oxygen or nitrogen oxides) simultaneously exist.

It grows over a wide range of pH values and temperatures.

It can be used in aquaculture ponds.

It can be used in agriculture to inoculate the phosphate fertilizers so as to enhance the absorption and bio

availability of P to the plants.

The role of Thiobacillus in controlling plant diseases in sulphur amended soils has been demonstrated with

regard to potato scab caused by Streptomyces scabies and the rot of sweet potatoes caused by S. ipomoea.

Under acidic soil conditions (below pH 5.0), inoculation of soil with thiobacilli after addition of sulphur

effectively minimizes losses due to these pathogens.

It has been used industrially in metal leaching from mineral ores and in the microbial desulfurization of coal in

combination with Thiobacillus ferrooxidans.

All sulfur-oxidizing bacteria use compounds such as H2S, S, and S2O32-

to generate energy using oxygen as the

terminal electron acceptor.

As usual, this is coupled to a proton gradient that generates ATP. The complete oxidation of these compounds

and the production of sulfuric acid (H2SO4) is common to many thiobacilli, which lowers the pH of their

surrounding environment.

Because many of the species of sulfur oxidizers produce sulfuric acid or ferric iron as an end product, they are

often associated with the corrosion of the environment in which they live.

Thiobacilli have been implicated in the destruction of pipes, concrete, and monuments.

They are also responsible for acid run-off from abandoned mines where the water leaving the mine can have pH

values as low as 2.

To add to the destruction, the acid dissolves minerals in the surrounding area leading to the solubilization of

many metals including aluminum, which can be toxic to aquatic organisms.

The high concentrations of iron and other metals along with the low pH make the water unfit for human

consumption or even for industrial purposes.

While the above activity of the thiobacilli can cause tremendous environmental harm, in controlled situations it

can have a very beneficial role in mining.

Many desirable metals exist in ores composed of highly insoluble sulfides and the concentration of the metal in

the ore is so low that it is not economically feasible to extract it by conventional chemical methods.

Under these conditions, microbial leaching, where a microbe is used to extract a desired chemical, is often

useful.

Leaching finds its greatest utility in the extraction of copper, but has also been employed to solubilize gold from

ores.

The ores attacked contain sulfur and Thiobacillus plays its role by using these reduced sufur compounds as

electron donors.

In the process the desired metal is released.

Microbial leaching of low grade copper ore is especially useful and serves as an example of the leaching process.

Cu2S can be acted upon by Thiobacillus ferrooxidans, which oxidizes Cu+ to Cu2+

, forming CuS and releasing

one Cu2+

ion.

The CuS then reacts with oxygen to form CuSO4.

The oxidation of CuS to CuSO4 can occur spontaneously, but T. ferrooxidans is also capable of performing this

conversion as part of its catabolism.

A third reaction relies of the fact that almost every ore contains iron pyrite (FeS2) and microbes will readily

attack this, releasing ferric iron (Fe3+

).

Once ferric iron is available it can react with CuS in a purely chemical reaction.

T. ferrooxidans further accelerates this process by using the oxidation of Fe2+

to Fe3+

as a method for energy

generation, thus keeping the concentration of ferric iron high.

The continual presence of ferric ions results in the release of large amounts of copper ions.

T thioxidans is an obligate chemolithotrophic bacterium which generates energy through oxidation of elemental

sulphur and reduced sulphur compounds such as thiosulphates and tetrathionates.

This bacterium is gram negative, rods, motile, non capsulated, non sporulating and aerobic.

T thiooxidans is an extreme acidophile and can grow at pH below 1 and the optimum pH is 3.5

S8 + 12O2 + 8H20 ---- T thiooxidan --- 8 H2SO4

T ferrooxidans is able to oxidise S apart from ferrous.

T thooxidans plays an important role along with T ferrooxisans in dissolution of metals from sulphidic ores.

Sulphur oxidation rate is an index of T thiooxidans indicating its ability to oxidize sulphur in the desired

conditions.

S - Oxidation is monitored interms of sulphate formed.

The sulphate is precipitated with barium chloride under controlled conditions to form barium sulphate crystals of

uniform size and the OD of the suspension is then measured.

A standard graph can be obtained by known concentration of sulphate per ml on OD at 420 nm Y-axis.

The amount of sulphate formed by T thiooxidans is then calculated from the graph.

13. Thiobacillus novellus

Synonyms: Starkeya novella

Thiobacillus novellus is a facultatively chemolithoautotrophic and methylotrophic, Gram-negative, rod-shaped

sulfur bacterium, shown by 16S rRNA gene sequence analysis to be a member of the alpha-2 subclass of the

Proteobacteria.

Starkeya novella (formerly Thiobacillus novellus) is obligately aerobic and oxidizer of sulfide and mercaptan,

It does not produce acid during oxidation and is facultatively able to degrade general BOD and a wide

assortment of fatty acids.

It is now recognized that the iron - oxidizing thi0bacillii can grow and metabolize iron, sulfur, or glucose.

REMOVAL OF H2S:

The results indicated that the Thiobacillus novellus biofilter under mixotrophic conditions possessed broad

adaptability of pH (5 ‐ 10) compared to the one under autotrophic conditions.

In a batch experiment high H2S concentration (>140 ppm) inhibited the enzymatic activity, thus the H2S removal

was limited by the reaction rate rather than the diffusion rate at diluted H2S concentration.

During the environmental shock experiment, 99.5% of the H2S content from the inlet was eliminated in a

mixotrophic environment, but only 97.5% was eliminated in an autotrophic environment.

Moreover, the mixotrophic biofilter possessed higher removal capacity than the autotrophic biofilter.

Thus, the results suggest that the potential of Thiobacillus novellus CH 3 in the mixotrophic environment should

be of selective advantage over the cells in the autotrophic environment.

(http://www.tandfonline.com/doi/abs/10.1080/10934529709376619)

OXIDATION OF THIOSULPHATE:

The pathway of thiosulfate oxidation in the facultatively chemolithotrophic, sulfur-oxidizing bacterium Starkeya

novella (formerly Thiobacillus novellus) has not been established beyond doubt.

Recently, isolation of the sorAB genes, which encode a soluble sulfite:cytochrome c oxidoreductase, has been

reported, indicating that a thiosulfate-oxidizing pathway not involving a multienzyme complex may exist in this

organism.

Here we report the cloning and sequencing of the soxBCD genes from S. novella, which are closely related to the

corresponding genes encoding the thiosulfate-oxidizing multienzyme complex from Paracoccus pantotrophus.

These findings suggest two distinct pathways for thiosulfate oxidation in S. novella.

The expression of sorAB and soxC in cells grown on thiosulfate- and/or glucose-containing media was studied by

Western blot analysis.

The results showed that the SorAB protein is synthesized in the presence of thiosulfate irrespective of the

presence of glucose.

In contrast, the SoxC protein is subject to repression by glucose; the repression, however, appears to be

dependent on the relative amounts of glucose and thiosulfate present.

The regulatory effects observed for the expression of sorAB are likely to be mediated by an extracytoplasmic

function sigma factor encoded by the sigE gene identified upstream of sorAB.

(http://link.springer.com/article/10.1007%2Fs002030000241)

SULFITE OXIDASE ACTIVITY

Thiobacillus novellus shows a maximum induction of sulfite oxidase activity and a maximum growth rate as a

result of supplementing the autotrophic growth medium with 4.0 microM ammonium molybdate.

Cells grown in the presence of molybdate showed approximately 10-fold increases in the amount of enzyme-

associated molybdenum and in the sulfite-to-cytochrome c and sulfite-to-ferricyanide reductase activities.

The effect of exogenous molybdate was not discernible with cells grown in the absence of thiosulfate.

Tungsten inhibited the growth of T. novellus and the expression of sulfite oxidase activity.

(https://www.researchgate.net/publication/16566486_Sulfite_oxidase_activity_in_Thiobacillus_novellus)

IN AGRICULTURE:

Sulphur oxidizers are involved in oxidation of elemental sulphur to plant available sulphate.

Sulphur is the key element for higher pulse production and plays an important role in the formation of proteins,

vitamins and enzymes.

Sulphur is a constituent of the essential amino acids cystine, cysteine and methionine.

In the recent years the importance of sulphur in pulse nutrition has been well recognized because of the

widespread occurrence of its deficiency in the soil world over.

Majority of sulphur taken up by plant roots is in the form of sulphate (SO), which undergoes a series of

transformation prior to its incorporation into the original 4 compounds.

The soil microbial biomass is the key driving force behind all sulphur transformations.

The biomass acts as both a source and sink for inorganic sulphate.

They make available sulphate from element or any reduced forms of sulphur, through oxidation process in the

soil.

The role of chemolithotrophic bacteria of the genus Thiobacillus in this process is usually emphasized.

http://link.springer.com/article/10.1007%2Fs002030000241

https://www.researchgate.net/publication/16566486_Sulfite_oxidase_activity_in_Thiobacillus_novellus

Use of sulphur oxidizers enhance the natural oxidation and speed up the production of sulphates.

14. Corynebacterium glutamicum

Synonyms:

Brevibacterium flavum

Micrococcus glutamicus" Kinoshita et al. 1958

Brevibacterium lactofermentum'

Corynebacterium lactofermentum'

Arthrobacter sp. NCIB 9666

Brevibacterium chang-fua

The microorganism is a nonpathogenic, nontoxigenic species and strain (Corynebacterium glutamicum) commonly used

in food processing.

1. Morphological properties

Gram stain: positive

Several characteristics of C. glutamicum makes it useful in biotechnology.

It is not pathogenic, does not form spores, grows quickly, has relatively few growth requirements, has no

extracellular protease secretion, and has a relatively stable genome (4).

C. glutamicum produces several useful compounds and enzymes. It was first discovered as a producer of

glutamate. Now it is also used to make amino acids, such as lysine, threonine, and isoleucine, as well as vitamins

like pantothenate (2).

Another possible use for C. glutamicum is in bioremediation, such as for arsenic. C. glutamicum contains two

operons in its genome, the ars1 and ars2 operons, that are resistant to arsenic. With further experimentation,

researchers hope to be able to eventually use this bacterium to take up the arsenic in the environment(4).

Current Research

Because of the useful characterisitics of Corynebacterium, much research has been done on it to try to modify it

in some way in order to make it more useful for humans.

One such way is by creating a biosynthetic pathway to produce poly(3-hydroxybutyrate) (P(3HB)), a polyester

that can be used to make a biodegradable plastic.

Plasmids were inserted into c. glutamicum, including an expression plasmid, that under certain conditions would

create the P(3HB).

This experiment was also done with E. coli.

The results showed that although the P(3HB)s created differed slightly in properties and although E. coli had a

higher P(3HB) content, C. glutamicum had almost four times higher cell density, making it a more efficient

producer of the polyester.

Further research will fine-tune the process as well as try to change the properties of the synthesized polyester

even more. (5).

Another area of research into C. glutamicum is how to increase its production of L-glutamic acid, which is

produced annually at a rate of about 1.5 million tons.

One way is to get rid of CO2, which would increase the production of L-glutamic acid.

To do so, phosphoketolase was used in order to bypass a pathway in which CO2 would normally have been

synthesized.

The yield of L-glutamic acid was increased by 9% by weight and productivity increased by 10%, implying that

the technique was a success.

This technique could be further developed, as well as transferred to increase production of other C. glutamicum

products, such as other amino acids. (6)

In a related area of research, another group tried to increase L-serine production in C. glutamicum.

Since serine has pharmaceutical uses and about 300 tons are produced annually, there is a lot of interest in

researching its production.

One of the intracellular processes that inhibits L-serine production involves an enzyme called serine

hydroxymethyltransferase (SHMT), whose activity can be controlled by 5,6,7,8-tetrahydrofolate (THF).

By deleting the genes for THF synthesis, an external folate source was needed for the growth of C. glutamicum,

but SHMT use was controlled.

This led to a higher yield of L-serine.(7)

References

1. Rollins, David M. "Pathogenic Microbiology." 2000.

http://www.life.umd.edu/classroom/bsci424/PathogenDescriptions/Corynebacterium.htm

2. Kalinowski, Jörn, Dr. "Fermentative Production of Amino Acids and Vitamins by Corynebacteria".

Universität Bielefeld. Genetik. http://www.genetik.uni-bielefeld.de/Genetik/coryne/coryne.eng.html

3. Genomic Sequence of Corynebacterium glutamicum. NCBI Database,

http://www.ncbi.nlm.nih.gov/sites/entrez?db=genome&cmd=Retrieve&dopt=Overview&list_uids=20857

4. Mateos, Luis M., Efren Ordonez, Michal Letek, and Jose A. Gil. "Corynebacterium glutamicum as a model

bacterium for the bioremediation of arsenic". International Microbiology. 2006. p. 207-215.

5. Jo, Sung-Jin, Michihisa Maeda, Toshihiko Ooi and Seiichi Taguchi. “Production System for Biodegradable

Polyester Polyhydroxybutyrate by Corynebacterium glutamicum”. Journal of Bioscience and

Bioengineering, Vol. 102, 233-236 (2006).

6. Chinen, Akito, Yuri I. Kozlov, Yoshihiko Hara, Hiroshi Izui and Hisashi Yasueda: “Innovative Metabolic

Pathway Design for Efficient L-Glutamate Production by Suppressing CO2 Emission”. Journal of

Bioscience and Bioengineering, Vol. 103, 262-269 (2007) .

7. Stolz, Michael, Petra Peters-Wendisch, Helga Etterich, Tanja Gerharz, Robert Faurie, Hermann Sahm,

Holger Fersterra, and Lothar Eggeling. "Reduced Folate Supply as a Key to Enhanced L-Serine Production

by Corynebacterium glutamicum." Applied and Environmental Microbiology, February 2007, p. 750-755,

Vol. 73, No. 3

15.Penicillium citrinum

Synonynms: P. steckii is an obsolete synonym of this species.

It contributes to manganese oxidation mostly by the production of acids and by changing the pH of the soil.

Reduction of manganese by bacteria is an indirect process.

For example by decreasing the pH, lowering of the O-R potential and removal of oxygen as a result of microbial

activity the level of exchangable manganese in the soil has been found to increase.

In some cases, MnO2 serves as an electron acceptor in respiration (RH2+MnOa--+Mn(OH)2+R).

Immobilization of manganese is not much of importance since microbial cells do not contain more than 0.05 per

cent of the element and this would not affect the process of oxidation or reduction.

There is however, no doubt that there is a manganese cycle in the soil involving the divalent and tetravalent and

other oxidation states of the element.

Mode of action

It appears that the microbial contribution to manganese oxidation is mostly by the production of acids and by

changing the pH of the soil.

Reduction of manganese by bacteria is an indirect process.

For example by decreasing the pH, lowering of the O-R potential and removal of oxygen as a result of microbial

activity the level of exchangable manganese in the soil has been found to increase.

In some organisms, MnO2 serves as an electron acceptor in respiration (RH2+MnOa--+Mn(OH)2+R).

Immobilization of manganese is not much of importance since microbial cells do not contain more than 0.05 per

cent of the element and this would not affect the process of oxidation or reduction.

There is however, no doubt that there is a manganese cycle in the soil involving the divalent and tetravalent and

other oxidation states of the element.

http://www.life.umd.edu/classroom/bsci424/PathogenDescriptions/Corynebacterium.htm

http://www.genetik.uni-bielefeld.de/Genetik/coryne/coryne.eng.html

http://www.ncbi.nlm.nih.gov/sites/entrez?db=genome&cmd=Retrieve&dopt=Overview&list_uids=20857

MEVASTATIN

MnO2 + 2H+ → Mn2

+ + 1/2O2 + H2O


Manganese is an essential micronutrient for the growth of plants, animals and microorganisms and occurs in the

soil in the tetravalent (Mn+4) or divalent (Mn+2) form while only the latter is utilized by the plants and

microorganisms.

Manganese availability in soils increases as soil pH decreases

A considerable portion of manganese like iron may also be bound in organic complexes.

Manganese can also undergo autooxidation depending upon the pH.

The active organisms in manganese oxidation include species of Arthrobacter, Bacillus, Corynebacterium,

Klebsiella, Pseudomonas and the fungi such as Cladosporium, Curvularia and Fusarium.

Penicillium Citrinum and Carnobacterium spp. are found to be more effective.

The plant growth promoting ability of this fungal strain may help in conservation and revegetation of the rapidly

eroding sand dune flora.

Penicillium citrinum is already known for producing mycotoxin citrinin and cellulose digesting enzymes like

cellulase and endoglucanase, as well as xylulase.

Gibberellins producing ability of this fungus and the discovery about the presence of GA5 will open new aspects

of research and investigations

.

15. Williopsis saturnus

The genus Williopsis was established by Zender (1925), based mainly on the formation of saturn-shaped

ascospores.

Later, the use of Williopsis was discarded, the nitrate-assimilating species of the genus were assigned to

Hansenula, and other Williopsis species were placed to Pichia (Wickerham, 1951; Lodder and Kreger-van Rij,

1952; Lodder, 1970; Kreger-van, 1984). von Arx et al. (1977) transferred the saturn-spored nitrate-assimilating

species from Hansenula to the reinstated genus Williopsis by hybridization studies.

Among the Williopsis species, W. saturmus, W. mrakii, W. suaveolens, W. subsufficiens, and W. sargentensis

shared 32–78 % total DNA homology based on the nuclear DNA-DNA relatedness (Kurtzman, 1991), and were

reassigned at the variety level.

Currently, five species, W. californica, W. mucosa, W. pratensis, W. salicorniae, and W. saturnus including five

varieties were accepted in the genus Williopsis (Kurtzman, 1998).

While, Williopsis revealed polyphytic and heterogeneous in rRNA sequence analysis, PCR finger printing and

karyotypes (Kurtzman and Robnett, 1998; Yamada et al., 1994, 1995; Naumova et al., 2004).

cell type: Asci with ascospores

Synonyms:

Hansenula beijerinckii, Hansenula coprophila, Hansenula mrakii,

Hansenula saturnus var. saturnus, Hansenula saturnus var. subsufficiens,

Hansenula suaveolens,Kloeckera saturnus, Pichia sargentensis, Pichia suaveolens,

Saccharomyces saturnus, Willia saturnus, Williopsis beijerinckii, Williopsis mrakii,

Williopsis sargentensis, Williopsis suaveolens, Williopsis subsufficiens

Colonies after 3 days on YPD Agar

CITATIONS:

A plant-growth-promoting isolate of the yeast Williopsis saturnus endophytic in maize roots was found to be

capable of producing indole-3-acetic acid (IAA) and indole-3-pyruvic acid (IPYA) in vitro in a chemically

defined medium.

It was selected from among 24 endophytic yeasts isolated from surface-disinfested maize roots and evaluated for

their potential to produce IAA and to promote maize growth under gnotobiotic and glasshouse conditions.

The addition of l-tryptophan (L-TRP), as a precursor for auxins, to the medium inoculated with W. saturnus

enhanced the production of IAA and IPYA severalfold compared to an L-TRP-non-amended medium.

The introduction of W. saturnus to maize seedlings by the pruned-root dip method significantly (P<0.05)

enhanced the growth of maize plants grown under gnotobiotic and glasshouse conditions in a soil amended with

or without L-TRP.

increases in the dry weights and lengths of roots and shoots and also in the significant (P<0.05) increases in the

levels of this was evident from the in plants IAA and IPYA compared with control plants grown in L-TRP-

amended or non-amended soil.

The plant growth promotion by W. saturnus was most pronounced in the presence of L-TRP as soil amendment

compared to seedlings inoculated with W. saturnus and grown in soil not amended with L-TRP.

In the glasshouse test, W. saturnus was recovered from inside the root at all samplings, up to 8 weeks after

inoculation, indicating that the roots of healthy maize may be a habitat for the endophytic yeast.

An endophytic isolate of Rhodotorula glutinis that was incapable of producing detectable levels of IAA or IPYA

in vitro failed to increase the endogenous levels of IAA and IPYA and failed to promote plant growth compared

to W. saturnus, although colonization of maize root tissues by R. glutinis was similar to that of W. saturnus.

Both endophytic yeasts, W. saturnus and R. glutinis, were incapable of producing in vitro detectable levels of

gibberellic acid, isopentenyl adenine, isopentenyl adenoside or zeatin in their culture filtrates.

This study is the first published report to demonstrate the potential of an endophytic yeast to promote plant

growth.

This is also the first report of the production of auxins by yeasts endophytic in plant roots.

[Amr H. Nassar, Khaled A. El-Tarabily, Krishnapillai Sivasithamparam Promotion of plant growth by an auxin-

producing isolate of the yeast Williopsis saturnus endophytic in maize (Zea mays L.) roots; Biology and Fertility

of Soils; Volume 42, Issue 2 , pp 97-108]

The effects of different W. saturnus inoculum levels together with S. cerevisiae on fermentation and production

of volatile compounds were studied in Emir Grape must.

Monocultures of W. saturnus and S. cerevisiae were also used.

Inoculum level influenced the yeast growth, chemical composition and volatile compounds.

W. saturnus began to die off with an increase in ethanol levels.

The amounts of ethyl acetate and isoamyl acetate increased, but concentrations of 3-methylbutanol and ethanol

decreased with increasing inoculum level from 5x106 to 1x10

8 cells/mL of W. saturnus in mixed cultures.

Mono- and mixed culture fermentations inoculated with W. saturnus and S. cerevisiae yeasts did not form

undesirable levels of flavour compounds.

According to chemical composition and volatile compounds the differences between obtained wines were found

significant.

The addition of 5x106 cells/mL of W. saturnus appeared to a suitable inoculum level.

It could be said that W. saturnus yeast can be used in mixed starter cultures with S. cerevisiae.

(http://www.tandfonline.com/doi/abs/10.1080/08905436.2012.724038#.Uy-4LM6XGgU)

http://www.tandfonline.com/doi/abs/10.1080/08905436.2012.724038#.Uy-4LM6XGgU

The marine-derived Williopsis saturnus WC91-2 was found to produce very high killer toxin activity against the

pathogenic yeast Metschnikowia bicuspidata WCY isolated from the diseased crab.

It is interesting to observe that the purified β-1,3-glucanase from W. saturnus WC91-2 had no killer toxin

activity but could inhibit activity of the WC91-2 toxin produced by the same yeast.

In contrast, the WC91-2 toxin produced had no β-1,3-glucanase activity.

We found that the mechanisms of the inhibition may be that the β-1,3-glucanase competed for binding to β-1,3-

glucan on the sensitive yeast cell wall with the WC91-2 toxin, causing decrease in the amount of the WC91-2

toxin bound to β-1,3-glucan on the sensitive yeast cell wall and the activity of the WC91-2 toxin against the

sensitive yeast cells.

In order to make W. saturnus WC91-2 produce high activity of the WC91-2 toxin against the yeast disease in

crab, it is necessary to delete the gene encoding β-1,3-glucanase.

(http://link.springer.com/article/10.1007%2Fs10126-009-9243-9)

The effect of the yeast on stability of L. bulgaricus and L. rhamnosus varied with temperatures: no effect at 4 and

40 0 C, increasing effects from 10 to 30

0 C with enhanced lactobacilli survival by 102 to 107-fold.

The yeast enhanced L. bulgaricus and L. rhamnosus stability by approximately 106 to 107-fold in fermented

milks with 5 per cent w/v and 20 per cent w/v milk solids at 30 9 C.

Research limitations/implications – Use of live yeast has limitations.

The yeast must not ferment lactose and galactose, and fermentable sugars cannot be used as sweeteners to avoid

yeast growth.

Further understanding of the interaction between yeast and LAB may eliminate the need to add viable yeast.

Originality/value – Use of yeast to enhance stability of LAB and probiotics is a novel concept.

Addition of selected yeast could be an effective means of enhancing stability of LAB and probiotics in fermented

milks to extend shelf-life and to retain nutritional value.

(https://www.researchgate.net/publication/249362440_Enhancing_stability_of_lactic_acid_bacteria_and_probiot

ics_by_Williopsis_saturnus_var._saturnus_in_fermented_milks)

Spoilage due to yeast and mould growth is a major issue for yoghurt quality and shelf-life.

There is a need to develop natural alternatives to chemical preservation.

The purpose of this paper is to determine the effectiveness of mycocinogenic yeast Williopsis saturnus var.

saturnus as a biocontrol agent against spoilage yeasts and moulds in plain yoghurt.

Design/methodology/approach –

Yoghurts were prepared from reconstituted skim milk and were challenged with spoilage yeasts and moulds.

The treatment contained the added mycocinogenic yeast and the control without.

All yoghurts were incubated at 300 C.

Yeast and mould growth were determined by observing gas formation and mould colony occurrence at regular

intervals.

Findings – W. saturnus var. saturnus inhibited growth of lactose-fermenting and galactose-fermenting yeasts

(Candida kefir and Kluvyveromyces marxianus), and lactose non-fermenting but galactose fermenting yeasts

(strains of Saccharomyces cerevisiae and Saccharomyces bayanus).

The yeast also inhibited growth of dairy spoilage yeasts moulds including Byssochlamys, Eurotium and

Penicillium. W. Saturus is a bio control agent.

Research limitations/implications –

The inhibition of this mycocinogenic yeast against yeasts and moulds was dependent upon the concentration of

the latter.

Thus, hygiene and good manufacturing practice are essential in order to keep the contaminant load down and to

ensure the effectiveness of the mycocinogenic yeast.

Originality/value –

The use of mycocinogenic yeast to control spoilage yeasts and moulds in yoghurt is a novel approach with a

potential to minimise yoghurt spoilage and extend the shelf-life of yoghurt. [Shao Quan Liu, Marlene Tsao;

Biocontrol of spoilage yeasts and moulds by Williopsis saturnus var. saturnus in yoghurt; Nutrition & Food

Science 01/2010; 40(2):166-175.]

Liu and Tsao (2010a,b) reported that the use of Williopsis saturnus var. saturnus enhances the survival of the

two probiotic bacteria L. bulgaricus and L. rhamnosus in fermented milk.

The same group concluded that the use of yeast enhance the stability of probiotic bacteria in fermented milk

thereby extend the product shelf-life and retain nutritional value (Liu and Tsao, 2010a,b).

http://link.springer.com/article/10.1007%2Fs10126-009-9243-9

https://www.researchgate.net/publication/249362440_Enhancing_stability_of_lactic_acid_bacteria_and_probiotics_by_Williopsis_saturnus_var._saturnus_in_fermented_milks

https://www.researchgate.net/publication/249362440_Enhancing_stability_of_lactic_acid_bacteria_and_probiotics_by_Williopsis_saturnus_var._saturnus_in_fermented_milks

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3525881/#B107




REFERENCES:

Nassar, K. El-Tarabily, and K. Sivasithamparam, “Promotion of plant growth by an auxin-producing isolate of

the yeast Williopsis saturnus endophytic in maize (Zea mays L.) roots”, Biol. Fert. Soils, 42:97-108, 2005.

17. Glomus mosseae 1 x 102 cfu / gm

Product Class: Biological Inoculant

Production process

Soil-based pot culture is a common method for production of vesicular-arbuscular mycorrhizal (VAM) fungal

inoculums.

Recently, solution culture techniques such as nutrient film and aeroponics have been adapted for the production

of inocula of VAM fungi.

These methods provide an alternative to soil-based pot culture for mass production of clean, soilless VAM

inoculum.

Clean propagules, especially spores, are not only useful for inoculation, but are also essential for critical

physiological and genetic studies.

Both solution culture techniques provide well-colonized root inocula but results of sporulation experiments were

quite different.

In the nutrient film technique, sporulation was sparse, except with full-strength Hoagland nutrient solution, when

plants suffered manganese toxicity.

In contrast, the highly aerated rooting environment of aeroponic culture stimulates rapid and abundant

sporulation of the VAM fungi.

In an aeroponic culture, inoculations with Glomus mosseae (Nicol. & Gerd.) Gerd. resulted in root colonization

and sporulation superior to that previously reported for a soil-based pot culture (15, 15a).

Seedlings of Trifolium parvijorum (T . glomerutum, T . pratense, T . subterraneum, Dactylis glomerata, wheat

(l'riticum vulgare), cucumber (Cucumis sativus), (Ipomoea batatas (L.) Lam., and onion (Allium cepa) seedlings

can also be used) were sterilized in concentrated sulphuric acid, chilled, and germinated on agar plates, by a

technique described by Nutman (1949).

All other seeds were sterilized for 15 min. in 10 ppm ethyl mercury phosphate.

They were then rinsed in fifteen changes of sterile water, and were incubated at 15' on 0.75 % agar in water

(w/v) again.

After germination, seedlings were transferred to agar slopes in sterile test tubes, which were kept in a

greenhouse.

Culture medium:

All test plants were grown in a nitrogen-deficient inorganic salt medium formulated by Jensen (1942) and used

extensively in nodulation studies.

It contained g./l. of distilled water: 1, CaHPO2; 0.2, K2HPO,; 0-2, NaCl; 0.2, MgS04.7H20; 0.1, FeCl2; 7.5,

shredded agar.

The medium was adjusted to pH 6.3 before autoclaving at 120' for 20 min.

Inoculum.

An uncontaminated inoculum of germinated resting spores of Glomus mosseae was obtained as described by

Mosse, 1959 et. al.

Spores excised from the sporocarps were sterilized in two changes of a freshly prepared 2 yo (w/v) solution of

Chloramine T containing 200 ppm streptomycin.

After washing, 20-30 sterilized spores were transferred to a small cellophan disk, which was placed on a large

piece of sterile cellophan spread over a soil agar plate containing 0.2 g. soil dispersed in 5 ml. 0-75 % (w/v) agar

in water and containing 0.001 % crystal violet.

After incubation for 5 to 6 days at 20' the small cellophan disks, now covered with a network of anastomosing

germ tubes, were tested for freedom from bacterial contaminants by incubating them on nutrient agar for 24 hr.

They were also examined microscopically and any with fungal contaminants were discarded.

More than 90 % of the disks carried a pure inoculum, which was introduced, without disturbing the mycelial

network, by placing the small cellophan disks, with the spore-bearing surface uppermost, near to an actively

growing seedling root.

Seedlings were grown in a nonshaded greenhouse for 6 to 8 weeks after which roots were washed and trimmed

to a length of 6-8 cm.

Root colonization by VAM fungi was confirmed by the nondestructive autofluorescence method of Ames et al.

Aeroponic culture.

Colonized seedlings (20-30) were placed into aeroponic chambers in a non shaded greenhouse with a ca. 10-12

cm.

The apparatus of aeroponic chambers was adapted from Zobel et al.

Glomus mosseae was cultured from April to September with mean maximum and minimum temperatures of 37

and 25°C and a maximum photosynthetic photon flux density (PPFD) of 1,725 ,umol s-' m-2. Low-Pi (0.03 pug

g-1) dilute Hoagland nutrient solution was used in each chamber as described by Sylvia and Hubbell, with the

solution pH initially adjusted to 6.50 + 0.05 with 1 N NaOH.

Roots of four randomly selected plants from each chamber were harvested 14 days after seedlings had been

placed in the aeroponic chambers.

Successive harvests were continued thereafter at 2- or 3-week intervals up to 14 weeks.

At each harvest, only roots grown out of the original roots (6-8 cm) were collected; they were cut into segments.

These root segments were checked for sporulation under a dissecting microscope and then cleared in 10% KOH

and stained with 0.05% trypan blue.

The total root length and root length colonized by Glomus mosseae were estimated by a gridline intersect

method, and the total number of spores produced in each segment was recorded.

After each harvest, the final pH of the nutrient solution in the chambers was recorded and the solution was

changed.

Roots of the remaining seedlings were trimmed to a length of 12 to 15 cm to prevent growth into the nutrient

solution.

Analytical method

Quality Control

Serial dilution method has been used to determine the propgalues in each batch.

From the production after thorough mixing through random sampling a small quantity is taken by the quartering

method and tested for its propgalues in the concentration of10-8

to 1012.

Separate register is maintained for quality check details and materials are passed for packing only when the lot

contains 1 x104.

Meanwhile, once in a quarter, random samples from the production is taken and checked for its consistency in

quality at independent laboratories or co manufacturers as well as at the unit by the professional staff.

18 Trichoderma harzianum

Synonyms:

Sporotrichum narcissi Tochinai & Shimada, (1930)

Trichoderma lignorum var. narcissi (Tochinai & Shimada) Pidopl., (1953)

Trichoderma narcissi (Tochinai & Shimada) Tochinai & Shimada, (1931)

Chemical name (IUPAC): NA

CAS No.: 67892-31-3

Structural formula: NA

Molecular formula: NA

Molecular mass: NA

Appearance, Color, Odor and Physical state:

The strain belong to the species Trichoderma harzianum Rifai. It is an asexual fungal species that belongs to the Class

Deuteromycetes, Order Hyphomycetes, Family Moniliaceae.

Characteristics of the genus include: rapidly growing colonies bearing repeatedly branched conidiophores in tufts with

divergent, often irregularly bent, flask-shaped phialides. Conidia can be either smooth walled or roughened and are

usually green, but can be hyaline.

The conidia of Trichoderma harzianum species are 2.8-3.2 x 2.5-2.8 Fm and are smooth walled with a subglobose to

short oval shape. When grown on oatmeal agar at 20BC, colonies may reach over 9 cm in diameter. Optimum

temperature for growth is in the range of 150C – 35

0C.

The strain belong to the species Trichoderma harzianum Rifai. T. harzianum Rifai, and likely also ITEM 908, is

an asexual fungal species that belongs to the Class Deuteromycetes, Order Hyphomycetes, Family Moniliaceae.

Boiling point: NA

Melting point: NA

Vapor pressure: NA

Density NA

Solubility in water and organic solvents: NA

Viscosity (liquid form): NA

Hydrolysis: NA

Photolysis


Under normal sunlight Trichoderma harzianum does not change.

Half life (DT50)

Estimated half-life period in surface water is 5.5 weeks

PROLOGUE

Trichoderma harzianum is a fungus that is also used as a fungicide.

In soil, they frequently are the most prevalent culturable fungi. They also exist in many other diverse habitats.

Trichoderma spores germinate and colonize the soil immediately surrounding plant roots, living off the nutrients that all

plants exude from their roots. As it is fast growing it is capable of out-competing and displacing crop pathogens (i.e.

disease-causing organisms).

They are strongly mycoparasitic on Rhizoctonia solani and Pythium aphanidermatum.

They are also used to produce Xylanase Enzyme.

Production process:

Maintenance of the culture

The efficient Trichoderma harzianum strains, which possess excellent properties in the control of fungal diseases such

as Pythium, Rhizoctonia and Fusarium, as well as naturally stimulating the plant’s growth; used for the mass

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multiplication were maintained in the microbiology laboratory as slant cultures and as glycerol stocks and supplied to the

production department whenever

required.

Mass multiplication of the biofertilisers

After getting the suitable strains the production process was fine tuned. Starter cultures are screened for potential human

pathogens of concern and batches with contamination above regulatory levels are destroyed.

Trichoderma harzianum strains were cultured at 30 "C on a synthetic medium

(SM; Okon et al., 1973) containing (g per litre of distilled water): glucose, 15; MgS04.7H,0, 0.2; KH2P04, 0.9; KC1, 0.2;

NH,NO,, 1.0; Fez+, 0-002; Zn2+, 0.002; agar, 20 (in solid medium).

Preparation of the carrier material

Good carrier material should possess the following criteria:

• Locally available

• High organic matter content with good moisture retaining capacity (50 %)

• No toxic substance

• Easy to process and friable

The cured material was packed in polythene covers and sealed using an electric sealer.

The polythene bags were marked with the name of the product, name of the manufacturer, strain number, recommended

crops, method of inoculation, date of manufacture, expiry date, price and full address of the manufacturer.

Analytical method:

Quality Control Suspensions are plated on appropriate non-selective (potato dextrose agar, PDA) or selective media. Identification of the

particular strains can be done by amplification of distinct DNA sequences, sequence determination and subsequent

comparison with known sequences.

Spontaneous changes can be detected by microscopic and macroscopic observation of the growth of the fungus on

standard media and comparison with standard description of the species.

No relevant proteinaceous products are synthesized and secreted by T. harzianum.

Serial dilution method has been used to determine the Colony Forming Units (CFUs) in each batch. From the production

after thorough mixing through random sampling a small quantity is taken by the quartering method and tested for its

CFUs in the concentration of10^ -6 to 10^

-10. Separate register is maintained for quality check details and materials are

passed for packing only when the lot contains 1 x10^12.

Meanwhile, once in a quarter, random samples from the production is taken and checked for its consistency in quality at

independent laboratories or co manufacturers as well as at the unit by the professional staff.

REVIEW OF LITERATURE

The antagonistic activities of Trichoderma harzianum against several pathogenic fungi have been reported by many

workers

(Wells et al. 1972, Henis & Chet 1975, Backman & Rodrigues-Kabana 1975, Hadar et al.

1979, Elad et al. 1980).

Phaeoacremonium aleophilum and Fomitiporia mediterranea causes a chronic wood wasting disease of kiwifruit

(Actinidia deliciosa). Trichoderma harzianum found to redress the problem by stimulating the wound healing process.

(Neri, L.; Baraldi, R.; Osti, F. and Di Marco, S. (2008)

Effects of Trichoderma harzianum applications on fresh pruning wounds in Actinidia deliciosa for the protection

against pathogens associated with the “wood decay” of kiwifruit. Cultivating the Future Based on Science: 2nd

Conference of the International Society of Organic Agriculture Research ISOFAR, Modena, Italy, June 18-20, 2008.)

MODE OF ACTION IN AGRICULTURE:

Trichoderma harzianum readily colonizes plant roots and some strains are rhizosphere competent.

It attacks and parasitizes the pathogenic fungi.

It controls almost every pathogenic fungus.

It enhances the plant and root growth.

It forms various compounds which are inhibitory to the growth of crop pathogens.

It gains nutrition from other fungi and thus eliminates them by competition.

It has evolved numerous mechanisms for attack of the pathogenic fungi.

It stimulates the plants natural immune system, making the plant more resistant to infection by disease-

causing organisms.

Plant Growth

Trichoderma coils around, penetrates, and kills other fungi that are pathogenic (i.e. cause disease) to crops. It

can digest their cell walls.

Trichoderma harzianum can also enhance or stimulate plant growth and help buffer plants from extreme

conditions such as water logging, drought, pH, nutritional stress, etc.

Trichoderma harzianum improves root efficiency and nutrient uptake resulting from mycorrhizal-like

associations.

Trichoderma harzianum protects developing roots from naturally occurring sub-lethal pathogens, resulting in

more feeder roots and many more root hairs.

Trichoderma harzianum stimulates the production of several natural plant hormones such as gibberellins and

cytokinins, enhancing the growth of plants and even the rooting of cuttings. Seed germination is often also

improved.

Target PATHOGENS IN AGRICULTURE: Phytophtora spp., Aspergillus spp., Pythium spp., Rhizoctonia solani, Fusarium spp., Botrytis cinerea, Sclerotium

rolfsii, Sclerotinia spp. and Ustilogo spp, etc.

Toxicology Trichoderma species are widespread in the environment and are innocuous.

This microorganism is not known to be an endocrine disrupter.

Toxicity class WHO

Not Listed in PAN Bad Actors. (PAN Bad Actors are chemicals that are one or more of the following: highly acutely

toxic, cholinesterase inhibitor, known/probable carcinogen, known groundwater pollutant or known reproductive or

developmental toxicant. NOTE! Because there are no authoritative lists of Endocrine Disrupting (ED) chemicals, EDs

are not yet considered PAN Bad Actor chemicals.)

LD50 acute oral

Acute oral toxicity (mice):

The acute oral LD50 toxicity of extract from the Trichoderma strains was greater than 4,000 mg/kg after 1 and 14 days.

The acute oral LD50 toxicities of trichodermin (antibiotic) in mice were greater than 1,000 mg/kg.

LD50 acute dermal

The acute subcutaneous LD50 toxicities of trichodermin (antibiotic) in mice were greater than 500-1,000 mg/kg

A dermal application of 0.5g of Trichoderma harzianum at 5 x 109 CFU/g produced no dermal response in rabbits after a

4-hour exposure period.

When a single 1150-1570 mg/Kg dose of Trichoderma harzianum was applied dermally for a 24 hour exposure period to

rabbits, there were no clinical signs of toxicity and no effects on mortality or body weight nor any signs of dermal

irritation during the study. The available information indicates that dermal toxicity is not likely to occur at a higher dose.

LC50 acute inhalation

NA

Inhalation would be another route of exposure for mixer/loader applicators and possibly early-entry workers. Based on

the Toxicity Category II classification of the pulmonary study, the Agency has decided that pesticide handlers must wear

a dust/mist filtering respirator with the NIOSH prefix N-95, P-95 or R-95.

(http://www.epa.gov/opp00001/biopesticides/ingredients/tech_docs/tech_119200.htm)

Eye irritation

The two acute eye irritation studies conducted with the TGAI, indicate a potential for severe eye irritation, placing the

Technical Grade Active Ingredient in acute Toxicity Category I. In one study, a single dose of 0.1g of the active

ingredient (approximately 5 x 108 cfu) was used to treat 1 rabbit. The results indicated that the microbial pest control

agent (MPCA) TGAI, Trichoderma harzianum, has the potential to cause serious ocular damage. The active ingredient

was a severe eye irritant.

In another study a single dose of 0.1 g of the active ingredient was administered into the everted lower right eyelid of a

sentinel male rabbit. The results of this study indicated that a 3 minute, 180 ml saline rinse, applied 3 minutes post

dosing, had no ameliorating effect on the irritancy of the active ingredient. The adhesion of the TGAI to the conjunctivae

http://www.pesticideinfo.org/Docs/ref_toxicity7.html#BadActor

http://www.epa.gov/opp00001/biopesticides/ingredients/tech_docs/tech_119200.htm

remained a serious effect of treatment even after rinsing. It is not clear from this study whether the eye effects were due

to the active ingredient or the inert, because the product is manufactured by an integrated process.

However, another eye irritation study was conducted in which the test material, administered at guideline levels, was

mildly irritating to the eyes or in acute Toxicity Category III. Six male rabbits were treated with a single dose of 0.1 ml

(0.04 g) of test material into the everted lower right eyelid. The maximum average irritation score was determined to be

15.3 at 24 hours post dosing. There was no corneal involvement after 72 hours and ocular irritation was no longer present

after 7 days, equivalent to a mildly irritating rating. This study was considered acceptable and can be used for labeling of

the EP. Workers, who are most likely to be exposed to the pesticide during mixing/loading, application and post

application activities, are required to wear goggles to mitigate against potential eye irritation.


Skin irritation

Non-irritating

Skin Sensitization

Hypersensitivity study (Guinea pig):

When Trichoderma harzianum strain in physiological saline was applied in occluded dermal patches, it caused delayed

contact hypersensitivity in guinea-pigs.

Carcinogenicity

It is not carcinogenic

Ingredients not listed by ACGIH, IARC, NIOSH, NTR, or OSHA

Mutagenicity

Trichosetin, a novel tetramic acid antibiotic produced in dual culture of Trichoderma harzianum and Catharanthus

roseus callus, was found to be non-mutagenic before and after metabolic activation as shown in the results of rec assay

and micronucleus test, respectively. In the rec assay, 10, 100, and 1000 μg.mL-1 of trichosetin did not exhibit any

inhibition zones on both H17 rec+ and M45 rec- Bacillus subtilis strains. In the micronucleus test, 10, 50, and 100

mg.kg-1 of trichosetin did not significantly induce the formation of MPEs in mice. Therefore, trichosetin does not

possess direct DNA-damaging capacity and chromosome-breaking effect.

(http://philjournalsci.dost.gov.ph/vol138no2/pdfs/Mutagenicity%20potential%20of%20the%20novel%20drug.pdf)

Teratogenicity

Does not seem to have any teratogenic effects in experimental laboratory animals.

Effect on reproduction

Not Available

Residue and effect on human According to human health, Trichoderma harzianum is considered non-pathogenic.

It is not an apportunist. A few cases of infections in immunocompromised persons are known for other Trichoderma

species but not for T. harzianum.

Cholinesterase Inhibitors: No

Trichoderma harzianum do not grow at or near the body temperatures of mammals or birds.

During simulated use conditions, Trichoderma harzianum produces no known metabolites of environmental or health

concern.

Metabolise in animal and human body:

Not Available. No antibiotics are known to be accumulated by the strain.

Metabolise and degradation in plant, soil:

Peptaibols have so far not been possible to detect in soil, implicating that the substances are not persistent in soil.

Moreover this strain helps in biodegradation of some toxins and chemicals in the soil and make the soil more inhabitable

to plants.

To alleviate the stress of continuous cropping for cucumber continuous cropping (CCC) system, a beneficial fungus

Trichoderma harzianum was isolated and applied to soil to degrade allelochemicals exuded from cucumber plants in a

Rhizobox experiment. The following phenolic acids (PAs), classified as allelochemicals, were isolated and identified


http://philjournalsci.dost.gov.ph/vol138no2/pdfs/Mutagenicity%20potential%20of%20the%20novel%20drug.pdf

from cucumber rhizospheres: 4-hydroxybenzoic acid, vanillic acid, ferulic acid, benzoic acid, 3-phenylpropionic acid,

and cinnamic acid. Mixed PAs added in potato dextrose broth, each with 0.2 gram per liter, were completely degraded by

Trichoderma harzianum after 170 h of incubation. In Rhizobox experiments, inoculation of Trichoderma harzianum in

the CCC soil also degraded the PAs exuded from cucumber plant roots. This degradation was 88.8% for 4-

hydroxybenzoic acid, 90% for vanillic acid, 95% for benzoic acid, and 100% for ferulic acid, 3-phenylpropionic acid,

and cinnamic acid at 45 days after plantation. Simultaneously, a significant (p ≥ 0.05) decrease in the disease index of

Fusarium wilt and an increase in dry weights of cucumber plants were obtained in pot experiments by application of

Trichoderma harzianum. This was mostly attributed to degradation of PAs exuded from cucumber roots in CCC soil by

Trichoderma harzianum and alleviation of the allelopathic stress. Application of beneficial microorganisms, such as

Trichoderma harzianum that biodegrades allelochemicals, is a highly efficient way to resolve the problems associated

with continuous cropping system.

(Lihua Chen, Xingming Yang, Waseem Raza, Junhua Li, Yanxia Liu, Meihua Qiu, Fengge Zhang and Qirong Shen;

Trichoderma harzianum SQR-T037 rapidly degrades allelochemicals in rhizospheres of continuously cropped

cucumbers; Applied Microbiology and Biotechnology; Volume 89, Number 5, 1653-1663)

Residue data from other countries:

Not Available

Residue analytical method on crops:

Not Available. With the application of Trichoderma via soil irrigation exposure is only foreseen of the soil flora and

fauna.

Fatty tissue accumulation:

Not Available

Max Residue Limit (MRLs):

There is no Codex Maximum Residue Level (MRL) for Trichoderma harzianum spp.

Acceptable daily intake (ADI):

Since it has no food or feed uses, dietary risk is not expected.

Besides its low dietary toxicity, potential residues of the pesticide can be removed from food commodities by washing,

peeling, cooking and processing, to lower dietary exposure and risk.

Pre-harvest interval (PHI) (into finished product):

Not Available. No reports on Trichoderma metabolites entering the food chain are available.

Effect on environment Environment fate

Trichoderma species are naturally occurring in soils throughout the world. The proposed application sites and application

rates would not cause a detectable increase over naturally occurring background levels. There would be no increased

exposure to any non-target wildlife of ecological concern. The proposed uses do not pose a "may effect" situation to any

endangered or threatened animal or plant species.

Volatility:

Owing to it’s low volatility, evaporation from products containing it will be minimal.

Absorption in the soil:

Bio control agents either based on fungi or bacteria that contain the spores and mycelial fragments of a naturally

occurring antagonistic fungus like Trichoderma harzianum. In nature, this fungus occurs in environment throughout the

world. In the normal ambient environment conditions (soil, water and air condition) the spores germinate, and infect the

targeted pests/pathogens and sporulates again. At times of extreme environmental condition it protects itself by forming

an outer coat over it and remains intact under quiescent condition as spore for quite long time till the unfavourable

condition remains. When favourable condition returns and if the spore comes in contact with target pests/pathogens, it

attachés with the epidermal cuticle and start germinating- produces germ tube which later develops into mycelium which

penetrates inside the host and then sporulates finally. However, when the spores are exposed for a long time to the

extreme conditions prevailing in the environment like high temperature, high pH and the like, it dies.

Generally, in non rhizosphere soil, the amount of these organisms decreased from 105 propagules/g at depths of 0 to 2 cm

to 103 propagules /g after several months. However, the densities of organisms remained at 10

5 propagules /g in the inner

rhizosphere, demonstrating that rhizosphere soils are a potential reservoir for these micro organisms. As it is biological,

the question of any chemical transformation never arises,. No major transformation products were identified

http://www.springerlink.com/content/?Author=Lihua+Chen

http://www.springerlink.com/content/?Author=Xingming+Yang

http://www.springerlink.com/content/?Author=Waseem+Raza

http://www.springerlink.com/content/?Author=Junhua+Li

http://www.springerlink.com/content/?Author=Yanxia+Liu

http://www.springerlink.com/content/?Author=Meihua+Qiu

http://www.springerlink.com/content/?Author=Fengge+Zhang

http://www.springerlink.com/content/?Author=Qirong+Shen

http://www.springerlink.com/content/0175-7598/

http://www.springerlink.com/content/0175-7598/89/5/

Leaching:

Improves soil fertility. It increases, soil organic matter and water holding capacity.

The inner rhizosphere i.e. the soil / root interphase where plants, insects, pathogens interact which acts as a potential

reservoir for these microbes determine the efficacy, survivability, persistency, mobility and degradation. Further, spread

of the spores however, through the infected insects/pathogen may be possible. In fact, the spread of spores is a useful

thing in controlling the pest from establishment. These fungi/bacteria are target specific and not harmful to non target

organisms when it spreads

Hydrolysis:


Photolysis:


Under normal sunlight Trichoderma harzianum does not change.

Effect on non-target organisms:

The biodegradability insures that there is no environmental pollution

Trichoderma harzianum do not grow at or near the body temperatures of mammals or birds.

Certain Trichoderma species demonstrated larvicidal activity against the plant pests, bark beetles, Scolytus scolytus, and

Scolytus multistriatus. Published literatures imply that Trichoderma spp. may be useful biological control agents for

these plant pests, the bark beetles. However, during field application, the main problem is getting the fungi to the larvae

which are under the bark. The results of these studies indicate that it seems unlikely that the antagonistic fungi will

establish the type of infection that would spread throughout natural populations of bark beetles. The fungus appears to be

symbiotic with the striped bark beetle, and is associated with forest bark engraving beetle, Ips calligraphus, but not

found pathogenic in laboratory bioassay in the Philippines. Trichoderma harzianum strain has not been directly

implicated in these published findings.


Effect on bird, bee, wild animals:

The species Trichoderma does not grow at temperatures above 28 degrees C and is not capable of growth in warm

blooded animals or birds.

There are two studies on file for the mallard duck and the bobwhite quail (MRIDs 43809714 and 43809715 respectively).

These were rated as Supplemental (scientifically sound but not meeting Guideline requirements) because the testing was

done with nominal concentrations of the test substance. The studies were conducted according to section 8.1 Part B of the

European Communities Council Directive 91/414/EEC and Guideline 71-1 of the EPA Subdivision E, 1982 Pesticide

Assessment Guidelines and deviated significantly from the 1989 EPA Subdivision M Guideline. Nevertheless, these

avian oral LD50 tests performed with the mallard duck and bobwhite quail showed no treatment-related effects after

dosing the birds at 2000 mg/kg.

A non-guideline temperature growth study (MRID 44214301) was submitted to supplement the claim of no adverse

effects on avian species. It was reported as not meeting the requirements of 40 CFR Part 160 for Good Laboratory

Practices (GLP). This study did not conclusively demonstrate that the active ingredient cannot replicate at the basal body

temperatures of birds. It is not a required study and does not need to be duplicated.

The Agency considered all these studies in support of the guideline requirement. Given the natural and widespread

occurrence of T. harzianum and the lack of reported avian pathogenicity in the open literature, the Agency does not

anticipate any avian toxicity or pathogenicity from the proposed uses of this fungi.


Two studies were submitted to demonstrate the effects of T. harzianum on honeybees. The first honeybee dietary

toxicity/pathogenicity study (MRID 43809717) did not quantify the dose rates of T. harzianum, or verify the test

medium. Although this study showed no toxicity to adult honeybees, it was considered supplemental/unrepairable. The

results of the reported study do not aid in risk characterization or assessment of infectivity or pathogenicity to nontarget

organisms, since duration of the study was only 96 hours.

The second submission (MRID 44439301) indicated no infectivity or significant differences in honeybee hive health as

measured by (i) field bee longevity, (ii) brood size and pattern, and (ii) hive weight and overall vigor. The submitted

results summarized toxicity/pathogenicity to honeybee and compared untreated hives with those exposed to T. harzianum

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over a 30 day period. It is not possible to make an independent assessment of the exposure and risk to honeybees based

on this summary.

In the meanwhile, the Agency requirement to demonstrate a lack of pathogenicity to honeybees has been met by the

recorded observations that no evidence of Trichoderma harzianum strain growth or infection was seen during the hive

experiment. No fungus infected bees were found in the hives throughout the study.


Effect on fish, aquatics:

The proposed use patterns for the products would not expose aquatic wildlife to the fungi.

Trichoderma harzianum strain, was not toxic to D. magna at 1.2x103 cfu/ml over 10 days. While the lack of

pathogenicity by Trichoderma harzianum to Daphnia magna is evident from the submitted data, the study does not,

however, fulfill the guideline requirement of a 21 day pathogenicity study. Daphnid death due to a fungus infection

would be well apparent within the 10 day time period but is more likely to be detected during the 21 days if the study

was continued to comply with guideline requirements. The study was, therefore, rated as supplemental, or scientifically

sound but not meeting guideline requirements. However, it is sufficient to make an assessment that the fungus poses a

minimal risk to invertebrates for the proposed label use patterns. This conclusion is further supported by the natural

occurrence of Trichoderma harzianum without any known detrimental effects on aquatic invertebrate populations.


Effect on natural enemies:

It does not affect predators and parasitoids

The fungi Trichoderma are not pathogenic to plants or insects.

18 Trichoderma Viride

Synonyms:

Hypocrea rufa

Trichoderma cf. viride Lieckfeldt 1998

Trichoderma lignorum

Trichoderma sp.

Trichoderma gamsii

Trichoderma Viride Rifai

USE IN AGRICULTURE

Trichoderma viride is a potential antagonistic fungus which prevents the crops from several diseases

Trichoderma viride utilize the protoplasm as a source of food and multiply its spores so as the spores of the

pathogenic fungi are destroyed.

Trichoderma viride secrets cellulase and chitinase enzymes which react with cell wall of the disease causative

pathogenic fungi or bacteria and dissolve the same.

Trichoderma viride also secrets toxic substances such as Glyotoxin, Viridin and Trichodermin which destroys

the fungal pathogens.

Trichoderma viride suppress the population of pathogen through competition, antagonism, antibiosis, predation

and hyperparasitism.

Trichoderma produce both volatile and non-volatile antibiotics which are inhibitory to majority of the target

pathogen and also cause lysis of host hyphae through production of enzymes.



Used in control of seed rot and soil borne plant pathogens and plant root diseases caused by pests such as

Pythium, Rhizoctonia, Cylindrocladium, Fusarium and Thielaviopsis.

For use as commercial seed treatments; on cuttings; nursery drench; in greenhouses; in-furrow spray and soil

treatment; shade house and nursery crops; greenhouse and nursery planting mix amendment; greenhouse and

nursery drench; greenhouse foliar spray; greenhouse chemigation; home and garden drench; planter box (on-

site); transplant starter; potting soil; broadcast to established turf; new turf seedlings.

The pesticide can be used on all food/feed commodities except sugarcane, pechay (bok choy), rice, mushrooms,

kiwi, tobacco, barley, oats, lemon, apple, and chickpea. Not for use on aquatic crops.

19. Pseudomonas fluorescence

Use in Agriculture

Pseudomonas fluorescens is a plant growth promoting rhizo bacteria and is a widespread facultative parasite of

soil borne pathogenic bacteria, pathogenic fungi and plant parasitic nematodes.

It has been associated with nematode induced wilt suppressive soils as a natural control agent.

It is known to have considerable potential as a biological control agent for application to wilt sick soils infested

with pathogenic bacteria, pathogenic fungi and plant parasitic nematodes.

This bio-agent colonizes the root surface and also endophytic in nature.

Pseudomonas fIuorescens through different mechanisms suppress the plant pathogens.

They include antibiosis by producing antibiotics viz., pyrrolnitrin, pycocyanine, 2,4, Diacetyle phloroglucinol

(DAPG) and production of siderophores (fluorescent yellow green pigment), viz., pseudobactin which limits the

availability of iron necessary for the growth of pathogens.

Other important mechanisms include, production of lytic enzymes such as chitinases and ~-1,3 glucanases which

degrade chitin and glucan present in the cell wall of fungi, HCN production and degradation of toxin produced

by pathogen.

It is a natural antagonist of Erwinia amylovora, the bacterial pathogen that causes fire blight in apple and pear.

The suppression of Erwinia amylovora by Pseudomonas fluorescens is achieved through competition for the

same ecological niche, i.e. for nutrients and space on apple and pear flowers.

Possesses the capability to destroy the cell wall of the fungal pathogens and annihilate them.

Hydrogen cyanide and antibiotics such as Pycocyanin and Phenazine, secreted by P. fluorescens inhibit the

growth of disease causing pathogens.

Siderospores secreted by P. fluorescens chelate with iron in the soil, and make it difficult for the pathogens to

proliferate.

Several plant growth substances including gibberellins like compounds secreted by P. fluorescens contribute to

vigorous crop growth.

A number of strains of P. fluorescens suppress plant diseases by protecting the seeds and roots from fungal

infection. (O' Sullivan, D.B., and O'Gara, F. (1992)

Traits of fluorescent Pseudomonas spp. involved in supression of plant root pathogens. (Microbiol Rev 56: 662-

676).

Competitive exclusion of pathogens as the result of rapid colonization of the rhizosphere by P. fluorescens may

also be an important factor in disease control. (http://genome.jgi-psf.org/psefl/psefl.home.html)

Scope of application:

Pseudomonads also have great potential in agronomic applications, since they are prolific colonisers of plant

surfaces and represent a significant component of plant microflora.

Furthermore, they have been identified to possess traits that make them suitable as agents for biological pest

control (O’Sullivan and O’Gara, 1992).

These traits include an ability to produce antimicrobial molecules (antibiotics, antifungals and siderophores) and

a capacity to compete aggressively with other microorganisms for niches and to exclude phytopathogens.

Target Species:

Synonyms:

Bacillus fluorescens liquefaciens Flügge 1886

Bacillus fluorescens Trevisan 1889

Bacterium fluorescens (Trevisan 1889) Lehmann and Neumann 1896

Liquidomonas fluorescens (Trevisan 1889) Orla-Jensen 1909

Pseudomonas lemonnieri (Lasseur) Breed 1948

Pseudomonas schuylkilliensis Chester 1952

Pseudomonas washingtoniae (Pine) Elliott

http://genome.jgi-psf.org/psefl/psefl.home.html

Pythium spp.,

Phytophtora spp.,

Rhizoctonia solani,

Fusarium spp,

Botrytis cinerea,

Sclerotium spp.,

Sclerotinia sp. and

Ustilogo spp, etc.

Crops Recommended:

Chillies, Cotton, Cucumbers, Cut flowers, Eggplant, Ginger, Oil seeds, Orchards, Ornamentals, Potato, Pepper, Pulses,

Rice, Sugarcane, Tomatoes, Turmeric, Vegetables, Vineyards.

Occurrence:

It occurs in the nature in a wide range of soil types, climate, environment is distributed throughout the world.

The fungus colonizes the rhizosphere of a number of plant species.

1. Dextrose (microbial food)

Dextrose monohydrate is purified, crystalline D-glucose containing one molecule of water of crystallization per

molecule of D-glucose.

Dextrose Monohydrate is a simple sugar generated from the hydrolysis of starch.

Since corn (maize) crops are so abundant, the majority of Dextrose Monohydrate and other sweeteners are

typically generated from cornstarch.

India till now no GM Corn crops are grown.

Industry employs several methods to hydrolyze starch and generate dextrose in bulk.

Several processes of separating and crystallizing dextrose by repeated seeding, washing and crystallization were

developed during the 1800s and early 1900s,46 but these methods were time consuming and costly.

Today, commercial processes begin with the solution produced by liquefaction of the starch in a jet cooker.

The most common practices involve treating starch with enzymes called amylases (which are also naturally

occurring molecules), or treatment with acid.

These industrial processes mimic those that occur in nature.

In the human body, starch digestion begins in the mouth in the presence of the enzyme amylase (saliva).

As amylase hydrolyzes carbohydrates in the mouth, foods that contain starch (and other sugars) like bread, pasta,

potatoes, ice cream and baked goods are broken down into more simple sugars.

As this occurs, the food tastes increasingly sweeter... that's why we like foods containing Dextrose Monohydrate

so much.

As digestion continues, the food moves into the stomach, a highly acidic environment.

The acid produced by the stomach further hydrolyzes the food into the simplest carbohydrate monomers, one of

them being Dextrose (d-glucose).

By using nature as a guide, industry generates large quantities of Dextrose Monohydrate from available natural

resources (i.e. corn)

(http://www.cooperativepurchasers.com/Ingredients/Dextrose/How-Is-Dextrose-Monohydrate-Made.html)

Saccharification produces a 94% dextrose liquor, which can be processed in several ways.

To make a dextrose syrup, the fats and protein are removed from this liquor, as given below.

Using immobilized enzyme technology, it is possible to produce high-fructose syrups containing 42%, 55% or

90% fructose.

A starch solution at about 35% solids and a pH of about 6.5 is drawn into a steam jet at 180°F (82°C) in the

presence of a calcium-stabilized, thermostable alpha amylase.

The slurry is maintained at this temperature through a series of loops for 3–5 minutes and then cooled to 95°C

(200°F) in a secondary reactor, where further alpha-amylase additions occur.

A holding time of up to 120 minutes in the secondary reactor produces a solution of approximately 12 DE. The

pH is adjusted to about 4.3 and gluco amylase is added.

Then the product is pumped to saccharification tanks where the enzyme reacts for 24–90 hours.

The gluco amylase reaction produces liquor containing 94% dextrose, which is then filtered to remove residual

protein and fats before being passed through beds of activated carbon, as was described for the corn syrup

process.

http://www.cooperativepurchasers.com/Ingredients/Dextrose/How-Is-Dextrose-Monohydrate-Made.html

Following carbon purification, the hydrolyzate is demineralized through anion and cation exchange resins prior

to being isomerized.

Considerable effort based on research work initiated in the 1950s resulted in enzyme technology able to convert

glucose to fructose on a commercial scale.

Current production of high-fructose syrups generally uses immobilized, rather than soluble, enzymes. Sources of

the enzyme include Streptomyces, Bacillus, Actinoplanes species.

The advantage of fixed bed systems is that the relatively high activity per unit weight allows manufacturers to

process large quantities of product through relatively small reactors in short times.

The short residence time in these reactors also reduces development of undesirable color and flavor compounds.

The reaction is essentially first order and reversible, following Michalis–Menten characteristics.

Proper demineralization of the liquor prior to isomerization is essential.

Depending on the source of the enzyme, optimum operating conditions include a pH range of 6.5 to 8.5 and a

temperature of 40–80°C.

Residence time in the reactor is usually less than four hours.

Enzyme decay is exponential; therefore the typical system will contain a number of reactors containing enzyme

in varying stages of output.

A typical half life of such a column may be as long as 200 days.

The fructose level of the output of each of these columns can be controlled by varying the reaction time (flow

rate), temperature and pH.

Once conversion is complete the liquor is pumped through beds of activated carbon and then evaporated to the

proper solids level, generally 71% or 80% dry solids as previously described.

The 42% fructose syrup from the isomerization column is first demineralized to remove trace components picked

up during isomerization, and is then pumped into the separator at 36–60% solids.

The relative difference in affinity of the resin for fructose and dextrose allows separation of the carbohydrates

into two enriched streams. As the 42% fructose feedstock is pumped through the bed, the fructose portion is

selectively absorbed relative to dextrose, resulting in separation of the two carbohydrates.

The syrup is then carbon bleached, demineralized and evaporated to 71% solids. The 94% dextrose liquid may

also be further refined to 99% dextrose by adsorption-separation chromatography prior to being bleached,

demineralized and evaporated.

Either anhydrous dextrose or dextrose monohydrate can be obtained by crystallization.

Monohydrate crystallizers are large horizontal, cylindrical batch tanks or continuous systems in which the crystal

mass is continuously removed, leaving about 20–25% of the batch to seed the next.

During crystallization, the syrup (75% solids, 95% dextrose) is cooled carefully and in a controlled manner

below 50°C.

Since crystallization of dextrose is an exothermic reaction, constant cooling is essential to maintain the proper

level of supersaturation.

When the magma of crystallized dextrose (�-D-glucopyranose) monohydrate is formed, the material is washed

and centrifuged in basket centrifuges to remove the mother liquor (‘first greens’).

The first greens may be reprocessed to yield a second crop of crystals.

The mother liquor from this step is known as ‘second greens’ or hydrol.

Both hydrol streams are combined to improve the yield.

The remaining monohydrate crystals are dried in a stream of hot air and packaged.

Documents

BIO SOIL MAGIC - Environmental XPRT · BIO SOIL MAGIC is intended for use in field crops, horticulture, turf, forage and ornaments. BIO SOIL MAGIC product should be used by persons