Role of microbes in Nitrogen cycle
Biogeochemical cycles – The chemical elements
of protoplasm tend to circulate in the biosphere
from environment to organisms and back to the
• Cyclic movement of Nitrogen from environment
to organism and back to the environment .
• One of the gaseous cycles in the ecosystem.
• Essential constituent of proteins, nucleic
acids, vitamins, coenzymes, alkaloids etc.
• Free Nitrogen of the atmosphere (79 % ) –
unavailable to plants.
• Microorganisms play important roles in
various processes of Nitrogen cycle such as
Nitrogen fixation, Ammonification,
Nitrification and Denitrification.
a ) Free living (non – symbiotic ) Nitrogen fixing
Live freely and independently in the soil.
Requires no host to carry out Nitrogen
E.g . Azotobacter, Azospirillum etc.
b ) Symbiotic Nitrogen fixing microbes -
Rhizobium, Bradyrhizobium, Sinorhizobium etc.
• Rhizobium is rod shaped, gram negative, motile
• They can grow either free - living in soil or can
infect leguminous plants .
• Rhizobium is host specific- infects and nodulates
specific host legumes - Pea, Beans, Soy bean
(Leguminosae) and establish a symbiotic
relationship - both partners are mutually
• Possess Nif gene for Nitrogen fixation - produce
enzyme Nitrogenase and red pigment
leghaemoglobin which protect the oxygen
sensitive Nitrogenase enzyme .
• Mature root nodule is the site of Nitrogen
• The atmospheric Nitrogen is reduced to
Ammonia and is released into the host plant
cells where it is assimilated into various
organic nitrogen compounds.
• Bacteria receive nutrients from the plant and
the plant in turn get nitrogenous compounds.
Symbiotic Nitrogen fixation by
• E.g. Nostoc, Anabaena etc.
fix atmospheric Nitrogen by
• Anabaena azollae establish
symbiotic relationship with
the water fern, Azolla which
is widely used to enrich
paddy fields with fixed
Anabaena – Azolla Symbiosis
• Fixed Nitrogen is absorbed by plants –
incorporated into proteins and other organic
• Animals depend plants either directly or
indirectly for their food – synthesize animal
proteins and other Nitrogen containing
• The dead organic matter of plants and animals are
degraded by ammonifying bacteria (Bacillus, Clostridium,
Proteus, Pseudomonas) and form Ammonia.
• Nitrifying bacteria oxidize Ammonia into nitrates. First,
Ammonia is oxidized to nitrite by bacteria such as
Nitrosomonas , Nitrosococcus etc. Nitrite is further
oxidised to nitrate by the bacteria Nitrobacter.
• Plants absorb Nitrogen mostly in the form of nitrates.
Some bacteria such as Pseudomonas denitrificans,
Thiobacillus denitrificans etc. reduce the nitrates of the
soil to gaseous Nitrogen. This results in the reduction of
fertility of the soil.
• Cultures of improved strains of nitrogen fixing
and phosphate solubilizing bacteria are used
• Stimulate plant growth by increasing the
availability of nutrients.
TYPES OF BIOFERTILIZERS
1. Nitrogen fixers
• Symbiotic nitrogen fixers
• Rhizobium leguminosarum – for Pea-Pigeonpea, Green
gram, Black gram, Cowpea; Groundnut, Soybean.
• R. phaseoli - for Beans
• R. japonicum - Soy bean
Cyanobacteria (Blue green algae)
Anabaena – Azolla symbionts - for upland and low land
Free - living (non –symbiotic ) nitrogen fixing
• Azotobacter (for Wheat, Maize, Cotton,
Potato, Mustard, Sugar cane)
• Azospirillum ( Wheat, Corn, Paddy,
Sugarcane, Sorghum ).
Frankia - can be used for reclamation of
2. Phosphorous Suppling Biofertilizers (PSB)
1. Phosphorous solubilizers or
Phosphobacteria - Solubilize different forms
of insoluble phosphates by producing citric
acid, succinic acid, fumaric acid etc.
e.g. Bacillus polymyxa
B. megatherium var. phosphaticum
3. Phosphorous mobilizers or Absorbers
• Vesicular Arbuscular Mycorrhizae (VAM) fungi
(Mycorrhiza – Association of fungi with the root system of
• VAM fungi colonize the root and increase the growth of
• Increase the uptake of Phosphorous which move slowly in
• Production of growth promoting substances
• Can increase yield to 30 -40%
• Better water holding capacity and tolerance of drought
4. Trichoderma as biofertilizer
• Selected strains of Trichoderma are used as biofertilizers -
Trichoderma harzianum is a good solubilizer of
• Enhances the uptake of water and nutrients , especially
Nitrogen, which leads to higher nutrient metabolism –
• Increases root and shoot growth.
• The plant also develops greater resistance to diseases ,
drought and salt.
• Reduces the activity of deleterious microorganisms in the
rhizosphere of plants.
• Colonize on the roots of the host plant, attack and kill the
soil pathogens – hence also used as a biocontrol agent.
Methods of Application of Biofertilizers
1. Seed treatment or Seed inoculation -
Biofertilizers like Azotobacter, Azospirillum,
Rhizobium etc. are used for seed treatment.
2. Dipping of seedlings – The seedlings are dipped
in the solution of biofertilizers before
transplanting. The treated seedlings are then
transplanted in the field . E.g. Azospirillum is
used for dipping of paddy seed lings.
3. Soil Application – The biofertilizers are mixed
with well dried, powdered Farm yard manure
(F.Y.M.). or compost and applied to the field
just before sowing of seed or transplanting of
Advantages of Biofertilizers
• Improve cycling of nutrients and increase soil
• Increase porosity and water holding capacity of
the soil and provide protection against drought.
• Enhance seed germination
• Increase crop yield to 20 – 30 %
• Cheap, convenient and ecofriendly
• Application of biofertilizers have considerable
importance in sustainable agriculture.
SINGLE CELL PROTEIN
• Protein derived from a culture of single-celled organisms, used
especially as a food supplement.
E.g. Cyanobacteria Spirulina (Spirulina maxima, Spirulina platensis
) and green alga Chlorella.
• Include microorganisms that are directly used as food source or as
a supplement . Earlier, it was known as ‘Microbial Protein’. The
term Single Cell Protein was coined by Scrimshaw (1967)
• SCP has high protein content, fats, carbohydrates, vitamins,
minerals etc. – the composition depends on the organism and the
substrate on which it grows.
• SCP can be used as a human food or cattle feed.
• Yeast, certain species of algae , fungi and bacteria can be grown
on suitable substrates and the cells are harvested as source of
Yeasts as SCP
• Widely used.
• Large scale production of yeasts is referred to as
microbial farming .
• The edible yeasts are known as food yeasts-
contain vitamins like Thiamine (B1), Riboflavin
(B2), Nicotinic acid (B3), Biotin ( Vitamin H, a
member of B –complex vitamins) etc.
• E.g. Saccharomyces cerevisiae (Baker’s Yeast),
Candida utilis (Torula yeast), C. guillerimondii, C.
tropicalis, C. lipolytica , Torulopsis candida, T.
utilis, Debaromyces etc.
Moulds as SCP
• Trichoderma reesei
• Fusarium venenatum has high protein content -
One of its strains is used commercially for the
production of SCP - mycoprotein Quorn.
• Rhizopus oligosporus
Cyanobacteria as SCP
• Spirulina maxima, Spirulina platensis etc.
• A most popular microbial food supplement.
• High protein content.
• Low calorie content.
• Used as a food source either as dried cake,
powdered product, tablets or capsules.
• Used as a ‘Slimming food’.
Bacteria as SCP
• Pruteen, produced from bacteria,
Methylophilus methylotrophus, cultured on
Methanol had 72% protein content, was the
first commercial SCP used as animal feed
• The bacteria Cellulomonas, Alcaligenes are
also used for the production of single cell
PRODUCTION OF SINGLE CELL PROTEIN
i) Selection of suitable strain of
ii) Selection of substrate
iii) Culture/ Fermentation
v) Post harvest treatment.
vi) Processing for use as SCP.
i) Selection of suitable strain of microorganisms
The quality of protein depends on the type of
microbial strain selected.
Strains of microorganisms for SCP production should be
selected based on
• their ability to produce protein of high quality
• capability to grow in ambient conditions
• ability to use a wide variety of substrates
• rapid growth rate
• with limited quantity of nucleic acid.
Such microbes are isolated from samples of soil, water,
air, biological materials etc.
ii) Selection of substrate
The substrate should be readily utilizable and cheap.
• Rice or wheat bran
• Fruit pulp
• Vegetable wastes
• Lignocellulosic agrowastes
• Whey from dairy industry
• Spent sulphite waste liquor derived after wood processing
• Methane rich natural gas
• Sewage etc.
iii) Culture/ Fermentation
• The pure culture of the selected strain of
microorganism can be cultured in tanks,
ponds(e.g. Spirulina) or in a fermenter or
bioreactor- deep lift fermenter, air lift fermenter
(e.g. Bacteria) in a suitable substrate under
optimum conditions of pH, temperature,
• The type of fermentation may be submerged (in
liquid medium), semi-solid (e.g. Cassava waste)
or solid state (e.g. rice or wheat bran)
• Microbes are cultured in continuous or fed-
Algal production for SCP
• Can be cultivated in
trenches, ponds etc.
• Require low intensity
• Temperature : 35 – 40
• pH range : 8 .5 – 10 .5
Image: Babul Aktar
Yeast production for SCP
• can be cultured on molasses, corn-steep
liquor, starch, pulp of fruits etc.
• Temperature : 30- 34 °C
• pH : 3.5 -4.5.
• The bulk of cells can be removed from the
fermenter by decantation.
v) Post harvest treatment
• Purification and drying should be simple –
centrifugation(yeast, bacteria), filtration
(filamentous fungi), washing, drying etc.
vi) Processing for use as SCP
a)Cell wall degradation -Liberation of cell
proteins by destruction of cell wall - either
by physical (Freezing –thawing, osmotic
shock, heating, drying),chemical (enzymes,
salts like sodium chloride, sodium dodecyl
sulphate) or mechanical (e.g. crushing,
crumbling, grinding, pressure
homogenization etc.) methods.
b) Reduction of nucleic acid content by
chemicals (acidified alcohol, salts etc.) and
• A fermented milk product.
• Curd can be used for direct consumption or for
the production of butter.
• Chemical composition of whole milk curd is
Water - 85 to 88 %
Fat - 5 to 8 %
Protein - 3.2 to 3.4 %
Lactose - 4.6 to 5.2 %
Ash - 0.7 to 7.2 %
Lactic acid - 0.5 to 1.1 %
PRODUCTION OF CURD
• Curd is formed from milk.
• Milk contains the sugar lactose and protein casein.
• Milk is inoculated with the starter culture and kept
for a few hours. Lactic acid bacteria
like Lactobacillus casei, Lactobacillus acidophilus,
Lactococcus lactis, Lactococcus cremoris,
Leuconostoc etc. grow in milk and ferment Lactose
sugar in milk into Lactic acid – results in
coagulation of milk protein casein to form the curd.
• Rennin (an enzyme from calf stomach , but now
produced by genetically engineered
microorganisms ) can also be used to promote curd
PRODUCTION OF VINEGAR
• A mixture of 4 % Acetic acid , small amounts of
alcohol, glycerol, esters, reducing sugars,
pentosans, salt and other substances .
• Composition of vinegar depend on the nature of
raw material used for the production of vinegar.
• Produced from fruit juices ( Apple, Grapes,
Orange etc.), starchy materials (Potato, sweet
potato) by heterofermentation- Alcoholic
fermentation by yeast, Saccharomyces
cerevisiae and acid fermentation by Acetobacter
TYPES OF VINEGAR
Depending on the raw materials , different
types of vinegar like
• Apple cider vinegar
• Wine vinegar
• Malt vinegar
• Balsamic Vinegar (from grapes)
• Rice Vinegar
• Cane Vinegar etc.
• Initially , the fruit juice is converted into ethyl
alcohol by alcoholic fermentation carried out
by yeast (Saccharomyces cerevisiae)
• Ethyl alcohol is then oxidized into acetic acid
by acetic acid bacteria, Acetobacter aceti,
Gluconobacter sps. etc. (Acid fermentation ).
Methods of production of Vinegar
1. Slow Methods
a) The Home or Let alone method
• Fruit juices or malt liquors undergo spontaneous
alcoholic and acid fermentations and form vinegar.
• A film of vinegar bacteria, Acetobacter aceti, called
‘mother of vinegar’ should grow on the surface of
liquid and oxidize the alcohol to acetic acid.
• Low yield because of the absence of productive
strains of vinegar bacteria
• Very slow method
• Vinegar of inferior quality.
b). The Orleans or French Method – for
continuous production of vinegar.
• Carried out in barrels
• Continuous fermentation process
• One – third of the barrel is filled with a good grade
vinegar as the starter culture – serves as the inoculum
of active vinegar bacteria, Acetobacter aceti
• Raw material (wine or malt vinegar ) is added to fill
the half of the barrel leaving an air space.
• The acetic acid bacteria growing in a film on top of
the liquid carry out the oxidation of alcohol to acetic
acid for weeks or months.
• Part of vinegar is drawn for bottling and is replaced by
equal amounts of wine or alcoholic liquor.
2. Quick Method - Generator Method
• Common method
• Gives high yield of good quality vinegar.
• Generator tanks made of wood
• Upper section – alcoholic liquor acidified with vinegar
is introduced with a sprinkling device (sparger)
• Middle section – the liquid is allowed to trickle down
over beechwood shavings or charcoal - which has
developed a slimy growth of acetic acid bacteria,
Acetobacter aceti – air enters through the false
perforated bottom - temperature is maintained at 29
to 30 °C - oxidise the alcohol to acetic acid.
• Bottom section – vinegar get collected.
Modern generators are
equipped with automatic
• feeding alcoholic liquid
• introduction of filtered air
• temperature control
• recirculation of liquid
collected at the bottom etc.
3. Submerged fermentation
Vinegar is produced in
Acetators by submerged
• allows strict control of
aeration and temperature.
USES OF VINEGAR
• In Pickling
• For preservation of meats
• For salad dressing.
Role of Microbes in Reconversion of Wastes
• Waste -unwanted and unusable materials which
is of no use - Solid wastes - Liquid wastes -
• Putrescible wastes – can be disposed by
microbial degradation - degraded by saprophytic
bacteria and fungi - The complex organic
molecules are broken down into simpler ones -
Biodegradable wastes .
• Non- Putrescible wastes– Not degraded by
saprophytic bacteria and fungi . These are
non - biodegradable wastes .
1. Solid wastes :
Undesirable solid or semisolid
Municipal solid wastes
The overall garbage created
by a community- include
• vegetable markets
• fish markets
• butcher shops
• schools and other
Municipal solid wastes
Hazardous solid wastes
• Highly dangerous wastes -
toxic, explosive or
corrosive and may cause
potential hazard to human
health and environment .
• Can not be handled, stored
, transported, treated or
disposed off without
1. Sanitory land filling
• Very common method
• Landfills represent an environmentally acceptable
disposal method of municipal solid waste on ground.
• The burial of solid waste in a landfill initiates a complex
series of chemical and biological reactions .
• The anaerobic biodegradation of solid waste requires the
coordinated activity of several groups of
• The predominant organisms participating in the
degradation process of municipal solid wastes are
members of the class Clostridia, Fibrobacter, Arcobacter,
Pseudomonas, Acinetobacter etc.
• Solid wastes are dumped in successive layers, one above
another, in low – lying land areas.
• Each layer is nearly 1.5 m thick - covered by 20 – 25 cm
• Keep it for 1 week – fill the next layer
• Waste collection vehicles compress waste so that more of it
can be stored in the same space.
• Spraying insecticide will reduce mosquitoes, flies etc.
• Whole waste get stabilized in a few months and settles
down by 20 – 40% of its original thickness.
• Hydrolysis of complex organic matter may occur under
anaerobic conditions , releasing CO2, CH4, H2S and other
gases and produce simple, water – soluble organic acids.
• Composting is the natural process of
decomposing and recycling biodegradable
organic materials into a humus-rich material
called compost by the successive action of
Types of Composting
i) Aerobic composting
ii) Anaerobic composting
Types of Composting
1. Aerobic composting
• Decomposition of biodegradable wastes takes place in presence
• The wetted organic matter- mixed farm residues, leaves, food
wastes etc. are collected in a heap.
• Aerobic saprophytic bacteria are the most important
decomposers. (e.g. Bacillus subtilis , Pseudomonas sps.). Other
microorganisms like saprophytic fungi (vegetative vultures of
plant kingdom eg. Aspergillus fumigatus, Penicillium sps.,
Trichoderma sps. ) and actinomycetes.
• After a few weeks or months, these biodegradable wastes will
be transformed into a dark-brown or black humus material
a ) Trench composting
• Solid wastes are deposited in trenches (4 – 10 m
long, 2 – 3 m wide, 0.7 – 1.0 m deep ) in successive 15
cm thick layers sandwiched by 5 cm thick layers of
semi – liquid cattle dung, until the waste heap rises to
30 cm or more above the ground level.
• A 5 – 7.5 cm thick layer of soil is spread over the top
of the heap to prevent wind – blowing of the waste
and entry of insects.
• Decomposition of the wastes will be completed in
about 4 – 5 months and the humus – like compost is
formed which can be used as manure.
b) Open Air Composting
• Select an area free from water logging.
• Solid wastes are dumped on open ground as 5-
10 m long, 1 – 2 m wide and 0.5 – 1 m high piles.
• The top of each pile is covered with cattle dung.
• After a few weeks, the piles are turned upside
down for cooling and aeration.
• The wastes will be converted into compost in
about 4 – 6 weeks.
c) Open windrow
• Suitable method to
produce large quantity of
• The biodegradable
wastes are piled in long
• The rows of wastes are
turned frequently to give
ii) Anaerobic composting
• Biodegradable wastes are kept in the absence of
oxygen inside a sealed tank or digester.
• Decomposition of the wastes are carried out by
• In anaerobic composting, the temperature of the
wastes increase up to 70 °C within 7 days - This heat
persists for 2-3 weeks and help for anaerobic
decomposition of wastes and kill pathogenic
• Anaerobic decomposition of organic matter results in
the formation of several inorganic substances such as
ammonia, hydrogen sulphide, carbon dioxide, organic
• Humus – like compost will be formed in about 4 – 5
Biodegradable wastes are
converted into compost by
mechanical devises in
• Reception of wastes
• Segregation of wastes
• Shredding (Pulverization)
• Stabilization of the wastes
– takes place in 3 – 6 days.
• Preparation of the
stabilized mass for
Vermicomposting - The technique of speeding up
the process of composting by the use of
earthworms - the process of producing
• Earthworms eat the organic wastes and convert
it into worm castings or vermicompost. – Once
ingested by the worms , these organic materials
undergo physical and chemical breakdown in
• The worms secrete enzymes – proteases, lipases,
amylases, cellulases and chitinases – bring about rapid
biochemical conversion of the cellulosic and
proteinaceous materials in the variety of organic wastes-
municipal solid wastes, household garbages etc.
• Thus by the feeding and the casting activity the
earthworms convert organic wastes into vermicompost.
• Earthworms commonly used in vermicomposting include
• Eisenia foetida
• E. andrie
• Perionyx excavates
• P. sansibaricus
• Eudrillus eugeniae
• Lumbricus rubellus etc.
2. Liquid wastes
• Agricultural and industrial
operations and human
activities produce liquid
wastes - include any form of
liquid residue like sewage -
the waste water from various
sources which may contain
animal excreta, domestic
wastes, industrial wastes etc.
• The bacterium Sphaerotilus
natans , popularly known as
sewage fungus , play an
important role in the
degradation of organic
matter in sewage.
3. Industrial wastes
Microorganisms can be used as bioremediators.
Pesticides – Certain Microorganisms or Cell
extracts or enzymes of microbes can degrade
• In recent years, many scientists have enriched,
isolated, cultured and screened a lot of
microbial floras, such as bacteria, fungi,
actinomycetes, algae and other microbial strains
from the natural sewage or soil to degrade
• Microbes detoxify the wastes .
Bacteria for Pesticide degradation :
• Can degrade Aldrin , Chlorpyrifos , Coumaphos , Diazinon, Endosulfan ,Endrin
• Can degrade Chlorpyrifos , Coumaphos , DDT , Diazinon, Dieldrin , Endosulfan ,
Endrin, Glyphosate etc.
Alcaligenes & Flavobacterium
• Can degrade Chlorpyrifos, Endosulfan Diazinon , Glyphosate , Parathion
Klebsiella, Acinetobacter, Alcaligenes, Flavobacterium, and Bacillus subtilis
• Can degrade Endosulfan.
Actinomycetes for Pesticide degradation :
• Streptomyces alanosinicus, Streptoverticillium
album, Nocardia farcinia, Streptomyces atratus,
Nocardia vaccini, Nocardia amarae,
Micromonospora chalcea etc. can degrade
Aldrin, carbofuran, chlorpyrifos, diazinon, diuron
Algae for Pesticide degradation :
• Green microalga Chlamydomonas mexicana
can degrade Atrazin.
Fungi for Pesticide degradation :
• Aspergillus fumigatus, A. terreus,
Penicillium citrinum, Trichoderma harzianum
etc. can degrade chlorfenvinphos.
Disposal of Toxic heavy metals
• Wastes from thermal power stations , metal
refining industries, electroplating units,
sewage sludge, tanneries etc. contain toxic
• Heavy metals include Mercury (Hg), Lead
(Pb), Chromium (Cr), Arsenic (As), Zinc (Zn),
Cadmium (Cd), Uranium (U), Selenium (Se),
Silver (Ag), Gold (Au) and Nickel (Ni) - Heavy
metals such as As, Cr, Pb, Hg, Cd, U, etc. are
persistent components of industrial
• Pseudomonas fluorescens , Pseudomonas
aeruginosa , Arthrobacter viscous,
Citrobacter sps. etc. are used as
bioremediators for heavy metal pollution.
• Rhizopus orrhizus can accumulate Uranium
• Penicillium chrysogenum is used for the
removal of Radium.
• Yeast, Saccharomycetes cerevisiae can
accumulate Uranium from dilute metal
• Thiobacillus thiooxidans can bring about the
bioleaching of Cobalt, Nickel and Zinc from their
• T. ferroxidans can be used in the recovery of
Gold and Silver.
• Streptomyces flavomacrosporus is a multi metal
• A genetically engineered bacterium Deinococcus
radiodurans can be used as a bioremediator to
degrade toluene and mercury from highly
radioactive nuclear wastes.
Disposal of Petroleum products
Oil-degrading microorganisms include
Pseudomonas putida (used for cleaning an oil
spill in Texas, USA in 1990),
Marinobacter, Acinetobacter etc.