27

Chapter – II

REVIEW OF LITERATURE

28

The review of literature focuses on the following aspects: (1) Earthworms and

their gut flora (2) Nitrogen mineralization (3) Nitrification (4) Factors effecting

nitrification (5) Nitrification and plant growth promotion.

Vermiculture technology is emerging as an “environmentally sustainable”,

“economically viable” and “socially acceptable” technology all over the world.

Vermicomposting is the term given to the process of conversion of biodegradable

matter by earthworms into vermicast. In this process, the unavailable nutrients

contained in the organic matter are partly converted to more bioavailable forms. The

use of earthworms was known for ages as “waste managers” for efficient “composting

of food and farm wastes” and as “soil managers” for “fertility improvement” for

“farm production”. It is now being more scientifically and also commercially revived.

Vermicast is also believed to contain hormones and enzymes which are acquired

during the passage of the organic matter through the earthworm gut. The hormones

and enzymes are believed to stimulate plant growth and discourage plant pathogens.

All- in- all, the vermicast is believed to be an excellent organic fertilizer and soil

conditioner. Experiments coducted by Gajalakshmi and Abbasi (2002, 2004) confirm

the earlier reports that vermicompost has more beneficial impact on plants than the

compost.

Earthworms in Nitrogen Mineralization:

Among the soil invertebrates, earthworms play an essential role in carbon

turnover, nitrogen mineralization, soil formation, cellulose degradation and humus

accumulation etc,. In the upper soil horizon the earthworms generate mosaic

microzone by their activity (Römbke et al., 2005; Tiunov and Kuznetsova, 2000). The

earthworms do (i) penetrate soil by burrowing activity hence increasing aeration; (ii)

29

transfer soil and organic matter by casting; (iii) humiliate organic material as a first

step in organic matter breakdown (including cattle dung in meadows); (iv) change the

diversity and improve the activity of the microbial community by selective feeding.

Bearing in mind large numbers of these animals in the soil, one may consider their

significant effect on microbial population.

Earthworms are important but much neglected component of ecosystems.

Understanding the key role of earthworms in many biogeochemical cycles and in soil

development requires an understanding of gut microbial diversity and their influences

on soil and plant growth. Review of earthworm, highlights the key ecological

functions of earthworms in mineralization of organic matter.

Selection of Earthworm species for present study:

There are certain controversies about the recommendation of earthworms for

vermiculture in India. Species like Eisenia foetida and Eudrilus eugeniae are exotic

species; however the groups who promote indigenous earthworms discourage them.

Though, there are nearly 350 species of earthworms distributed in the country and

nearly 3000 species at the global level, Eisenia foetida is the universally accepted

species for vermicomposting.

E. foetida

E. foetida is ubiquitous endogeic species that is commonly found associated

with manure and has, therefore, earned the common name, manure worm. It is also

known as a red wiggler. It requires copious amounts of decaying organic matter and

can be found in decaying logs, compost and manure piles (Roberts and Dorough,

1985). Soil consumption for E. foetida is estimated to be 16 mg soil/ individual/ day.

Earthworms increase the amount of mineralized nitrogen from organic matter in soil.

30

The ratio of nitrate nitrogen to ammonium nitrogen tends to increase when earthworm

are present. (Ruz- Jerez et al.,1988). The nitrogen excretion rate of E. foetida has been

estimated at 0.4 mg/ g/ day, which is very high relative to other earthworm species

(Stafford and Edwards, 1985).

Eisenia foetida can survive in captivity under semi natural conditions, tolerant

to wide range of substrates and to other physical parameters like pH, temperature,

moisture and physical disturbances. These characters are found in very few species

of earthworms and hence, it is successful for culturing irrespective of their place of

origin (Kale, 1991).

To better understand the role of earthworms in nature and their potential

usage, the mechanisms of earthworm intestinal microorganisms must be studied.

However, little attention has been paid on the earthworm intestinal microorganisms of

E. foetida. Mechanisms of these microorganisms were not fully exploited or

elucidated.

It is known that earthworms cannot exist on pure microbial cultures but they

need mixed cultures of microbial species (Edwards and Bohlen, 1996). As food,

bacteria are of minor importance; algae are of moderate importance, fungi and to

lesser extent protozoa, are of major importance (Edwards and Fletcher, 1988). It is

generally accepted that earthworms do not commonly consume bacteria and

actinomycetes but they may proliferate in the process of passage through gut

(Kristufek et al., 1992). It was demonstrated that E. foetida cocoons were unable to

develop to maturity when only provided with fungi and bacteria but required protozoa

to develop to sexual maturity (Miles,1963). The microbial composition changes

31

qualitatively and quantitatively during passage through the earthworm intestine

(Pedersen and Hendriksen, 1993).

Karsten and Drake (1995) had found aerobic and anaerobic microorganisms to

be more numerous in the guts of both the epigeic (Lumbricus rubellus) and endogeic

species, (Octolasion lacteum) of earthworm than in the beech forest soils from which

they were obtained. The overall enumeration results indicate that microorganisms

capable of growth under anaerobic conditions were two to three orders of magnitude

in earthworm intestines than those from the soil where they were obtained. In

earthworms, the anaerobic microbial growth potentials were approximately equal to

aerobic microbial growth potentials, whereas, in soil samples, the aerobic microbial

growth potentials were much greater than anaerobic microbial growth potentials.

These results suggested that "the earthworm gut might constitute a micro-habitat

enriched with microbes capable of anaerobic growth and activity" in otherwise well

aerated soils.

Microorganisms in the gut of earthworms

The evidence of indigenous microorganisms associated with earthworms

comes from Jolly et al. (1993). Using scanning electron microscope, these researchers

had observed segmented, filamentous organisms attached to the hindgut epithelium of

Lumbricus terrestris by “socket-like” attachments.

Using light microscopy, Karsten and Drake (1995) had observed mixtures of

rods, cocci and spore forming microbes in earthworm gut homogenates with no single

dominant morphological type. There is strong evidence that microorganisms are able

to enter earthworm cocoons of Eisenia foetida from the soil in which they were

produced (Zachmann and Molina, 1993). Investigation of the interaction of

32

microorganisms with soil invertebrates is one of the ways to study the development in

biogeocenoses. At present the literature contains sufficient evidence of the presence of

bacteria, fungi and actinomycetes in the gut of soil invertebrates (Kalsen et al., 1992;

Fischer et al., 1995; Vincelas-Akpa and Loquet, 1995; Karsten and Drake 1995).

Mostly the population density of actinomycetes in the intestinal tract of invertebrates

was studied (Polyanskaya et al., 1996; Szabo, 1974; Chu et al., 1987). Toyota and

Kimura (2004) had studied widely on microbial community indigenous to the

earthworm Eisenia foetida.

Parle (1963) reported the presence of microbes in the intestine of earthworm

and an increase in the bacterial and actinomycetes poulations during passage of food

through the gut of Lumbricus terrestris. Bassalik (1913) showed that gut of

L.terrestris contained more culturable aerobic bacteria than soil.

Similarly, higher numbers of culturable aerobes were obtained from the guts of

Lumbricus terrestris, Allolobophora caliginosa and Allolobophora terrestris than

from soils (Parle 1963). However, several anaerobic nitrogen fixers Clostridium

butyricum, Clostridium beijerincki, Clostridium paraputrificum have been isolated

from E. foetida (Citernesi et al., 1977). The selective proliferation of microorganisms

i.e., fungi was reported in the gut of different kinds of earthworms like in P. millardi

(Gosh et al., 1989), Lumbricus mauritti and Eudrilus eugeniae (Parthasarathi and

Ranganathan, 1988), O. borincana (Alonso et al., 1999), bacteria in A. caliginosa

(Scheu, 1987), L. terrestris (Wolter and Scheu, 1999) and actinomycetes in L.

terrestris, Aporrectodea longa and A. caliginosa (Parle, 1963), L. rubellus and A.

caliginosa (Kristufek et al., 1993).

33

Earthworms not only disperse microorganisms important in food production

but also associated with mycorrhizae and other root symbionts, biocontrol agents and

microbial antagonists of plant pathogens as well as microorganisms that act as pests

(Edwards and Bohlen, 1996). Several researchers have demonstrated the ability of

earthworms to promote the dispersal of beneficial soil microorganisms through

castings, including pseudomonads, rhizobia and mycorrhizal fungi (Edwards and

Bohlen, 1996; Buckalew et al., 1982; Doube et al., 1994a; Doube et al., 1994b;

Madsen and Alexander, 1982; Reddell and Spain, 1991; Rouelle, 1983; Stephens et

al., 1994).

The role of microbial activity in the earthworm gut, cast and soil is very

essential for the degradation of organic wastes and the release of nutrients to plants.

During vermicomposting process when organic matter passes through the worm‟s gut,

it undergoes physico-chemical and biochemical changes by the combined effect of

earthworm and microbial activities. Vermicomposts/casts are coated with

mucopolysaccharides and enriched with nutrients. The cellulolytic, nitrifying and

nitrogen fixing microbes are found established in the worm cast (Kale et al., 1988).

Among the various types of microorganisms fungi, actinomycetes and bacteria are

predominant in vermicompost and soil. They play an important role in the

degradation of organic wastes. The earthworm burrows are lined with earthworm

casts which are excellent media for harbouring N-fixing bacteria (Bhole, 1992).

Presence of N-fixing bacteria in the gut content together with suitable conditions for

N-fixation also has been observed by Barois et al., (1987). According to Bhawalkar

(1993b) bacteria and fungi constituted the prime work force in soil. Earthworms

provide regulated micro-environment and the team of bacteria and earthworms was

considered to be the most derived work force in soil for speedy and effective bio-

34

processing of organic residues. Parthasaradhi and Ranganathan (1998) had reported

an increase of bacterial population in Lampito mauritti and Eudrilus euginiae worked

vermicompost. The increase of microbial population may be caused by the ingestion

of nutrient rich organic wastes that act as a substrate for the growth of microorganism

as reported by Tiwari et al., 1989. Further the total number of microorganisms in

earthworm gut content and casts depend on the quality of initial source of organic

matter/ soil, the greater the organic matter content the larger will be the microbial

population (Syers and Springett, 1984 and Edwards and Bohlen, 1996). Kale and

Bano (1992) had observed the mycorrhizal propagules in earthworm cast, which

survived upto 11 months, indicating the role of earthworms in the dissemination of

vesicular arbuscular mycorrhizal fungi. According to Indira et al., (1996) population

of beneficial organisms like phosphate solubilising bacteria, nitrogen fixing organisms

and entamophagous fungi was high in vermicompost. However, considerable

population of coliform bacteria was also noticed in vermicompost, which are known

to be hazardous to the health of persons handling the material.

Influence of Earthworms on Soil Nutrients:

Nitrogen is one of the most extensively distributed elements in nature i.e., up

to 78% in atmosphere. Of the total nitrogen found in nature, 99.96% is present in the

atmosphere, the chief reservoir. However, nitrogen is most often the limited nutrient

for crop production since only a fraction of atmospheric nitrogen is made accessible

to the plants through biological nitrogen fixation (Azam and Farooq, 2003). The term

nitrogen mineralization is most often used to describe the net accumulation of NH4+

plus NO3- and is thought to represent the amount of nitrogen available for plant

uptake. Mineralization of organic nitrogen is one of the main factors governing the

annual amount of manure applied to agricultural cropland. Earthworms directly cycle

35

the nitrogen by excretion in the casts, urine and mucoprotein and through the turnover

of earthworm tissues (Lee, 1985). Earthworm and other consumers have been shown

to stimulate nutrient cycling (Kitchell et al., 1979; Syers et al., 1979) through their

feeding and burrowing activities (Brown et al., 1978; Loquet et al., 1977). The

feeding activities of microinvertebrates have been shown to greatly enhance the rates

of decomposition of organic material in a variety of systems. In particular, Syers et

al., (1979) had shown increased rates of nitrogen mineralization in earthworm

castings. Kitchell et al., (1979) had discussed the importance of earthworms and other

invertebrates in translocating organic material and thus altering the availability of

nutrients and enhancing microbial mineralization processes. The earthworm output

comprises almost assimilable products of excretion such as ammonia and urea and

body tissues which are rapidly mineralized, thus it represents a potentially significant

source of readily available nutrients for plant growth (Satchell, 1967; Christensen,

1988).

Physical effects Biological effects

- Redistribution - Root distribution

- Soil penetratability - Microorganisms

- Ion movement

Fig 1: Physical and biological effects on soil nutrient supply which are influenced

by earthworms

POTENTIAL NUTRIENTS IN SOIL

PHYSICAL AND BIOLOGICAL EFFECTS OF

EARTHWORMS

NUTRIENTS TO

PLANTS

36

Earthworms have multiple, interactive effects on rates and patterns of

nitrogen mineralization and immobilization in natural and managed ecosystems

(Edwards and Lofty, 1977; Lee, 1983; Lavelle and Martin, 1992; Blair et al.,

1995b). Estimates of nitrogen mineralization by earthworms are 100 Kg ha-y

- for a

mixed deciduous forest (Satchell, 1963) and 150 Kg ha- y

- for a pasture (Keogh,

1979). Estimates of the direct effects of earthworms on nitrogen transformations

indicate that a significant amount of nitrogen fluxes through earthworm populations.

For instance, Parmelee and Crossley (1988) estimated an annual flux of 63 Kg

nitrogen ha-y

- through the earthworm community in a no tillage agroecosystem,

suggesting a significant effect on the transfer of nitrogen from litter to the soil organic

and mineral pools. Earthworm casts are enriched in terms of available nutrients and

microbial numbers and biomass, relative to the surrounding soil (Shaw and Pawluk,

1986; Lavelle and Martin, 1992). Other effects of earthworms on nitrogen cycling

processes are due to interactions between earthworms and the soil microbial biomass,

and are related to the influence of earthworms on the availability and storage of soil

organic resources (Lavelle and Martin, 1992). Barley and Jennings (1959), Scheu

(1987), Buse (1992), Haimi and Einbork (1992), Robinson et al., (1992). Hameed et

al., (1993) had reported increased N-mineralization due to earthworm activity in pot

experiments but few have shown how these effects have varied in response to

different nutrient inputs.

Scheu (1987) demonstrated that the effects of A.caliginosa on net nitrogen

mineralization were enhanced by upto 1.90 fold when old oak litter was added, but

were reduced to 65% when fresh oak litter was added or 25% when fresh ash litter

was added. Hameed et al., (1993) reported that L. terrestris increased nitrogen

mineralization when a high-quality litter (C:N=9.5) was added to the soil, compared

37

to low-quality litter (C:N=42.6). An increase in the rate of nitrification in casts

relative to the associated soil has also been suggested by several workers (Blanke and

Gieske, 1924; Day, 1950; Joshi and Kelkar, 1952), pointing to the potential

importance of earthworms in the N cycle. Although Parle (1963) observed that 96%

of the extractable inorganic N in fresh casts was present as NH4+ N while rapid

nitrification occurred following cast deposition. The above observations suggest that

the major participation of earthworms in the nitrogen cycle lies, directly or indirectly,

in their ability to increase the rate of mineralization of organic nitrogen.

Several workers have compared the chemical decomposition of worm casts

with soil. It has been claimed that nitrification is enhanced in casts. The casts contain

more nitrate, amino acid and total nitrogen than soil (Joshi and Kelkar, 1952; Day

1950). Wittich, 1953; Shrinkhande and Pathak, 1951; Nye 1955, had shown that when

worms were kept in pots all the nitrogen accounted for, their activity does not increase

total nitrogen of the system. However, it is evident from analyses of mineral nitrogen

in casts that worms change the character of ingested nitrogen and this is probably

important in the nitrogen cycle. Ammonia in fresh casts forms about 96% of the

extractable mineral nitrogen and this is rapidly converted to nitrate. The pattern of

nutrient release and microbial reorganization in fresh casts is similar to that observed

in temperate earthworms (Sharpley and Syers, 1979). The decrease of ammonium

concentration seems to be all the more rapid as temperature and clay activities are

high. Part of the ammonium is transformed into nitrates which are reorganized into

microbial biomass.

38

Nutrient value and plant growth promoting properties

Earthworms reject significant amounts of nutrients in their casts. In part these

losses result from the intense microbial activity in their gut, and from their own

metabolic activity. Eg. Elimination of N due to fast turnover of this element in

microbial biomass. A significant proportion of C assimilated by earthworms is

secreted as intestinal and cutaneous mucus with greater C:N ratios than those of the

resource used (Lavelle et al., 1983; Cortez and Bouche, 1987). As a result, part of the

nigtrogen assimilated may be in excess and have to be excreted. Another reason for

high mineral-N excretion is the rapid turnover of nitrogen in earthworm biomass as

shown by Ferriere and Bouche (1985) for temperate earthworms and further

confirmed for P. corethrurus, the pantropical species (Barois et al., 1987). The

nitrogen is mainly excreted as ammonium in the urine released in the gut, in case of

species which possess endonephridia and it is, thus, mixed with the soil and can be

found in the casts (Laverack, 1963).

Joshi and Kelkar (1952) reported that earthworm casts contained greater

percentage of finer fractions like silt and clay than in the surrounding soils. This

change in mechanical composition of soil was probably due to the grinding action of

earthworm gizzard. The chemical analysis of vermicasts revealed that they were

richer in soluble salts, neutral or alkaline in reaction and had higher percentage of

exchangeable Na, K and Mg but a lower exchangeable Ca than in corresponding soil.

Earthworm casts also contained greater amounts of Nitrogen (N), Phosphorous (P)

and Potassium (K). The vermicasts contained higher amounts of nitrate nitrogen and

possessed a greater nitrifying power than the corresponding soils.

39

Dash and Patra (1979) reported that vermicasts contained 0.47 percent

nitrogen compared to 0.35 percent nitrogen in the surrounding soil. Nitrogen

contribution from mucus, dead earthworm tissue and worm casts amounted to 180 Kg

ha-1

year-1

, whereas, Giraddi (1993) stated that vermicompost contained 0.8 percent

nitrogen, 1.1 percent P2O5 and 0.5 percent K2O, which was two, five and five times

respectively compared to that of FYM. Vermicompost also contained Mg, Ca, Fe, B,

Mo and Zn in addition to some of the plant growth promoters and beneficial

microflora. Bhawalkar and Bhawalkar (1993) reported that earthworms brought

several nutrient elements to the surface from deeper soil profile. Evidences showed

that concentrations of exchangeable Ca, Na, Mg, K and available P and Mo were

higher in earthworm casts than in the surrounding soil. In addition to the physical

mixing of soil by burrowing activities of earthworms, soil enrichment was achieved

by speeding up of mineralization of organic matter to an extent of 2-5 times. Several

valuable compounds were also produced through the earthworm – microfloral

interaction, which included vitamins such as B12 and plant growth hormones such as

gibberellins.

Barois et al., (1987) observed an activation of N mineralization, with the casts

having 270 percent more ammonia than the bulk soil. There was considerable increase

in available P2O5 and K2O in the earthworm treated organic wastes than that of

original and untreated waste according to Jambhekar (1992). Within a year of

application of vermiculture technology to the saline soil, 37 percent more N, 67

percent more P2O5 and 10 percent more K2O were recorded as compared to chemical

fertilizer (Phule, 1993). However, Shinde et al., (1992) opined that there was a need

to assess the additional merits of vermicompost, if any, over conventional

FYM/composts, because the overall nutrient status and the comparative performance

40

of the vermicomposts were in similar range of the conventional FYM. Kale et al.,

(1992) also felt that vermicompost was like any other organic manure, depending on

the nature of waste used as feed for worms, in which N, P and K level varied between

0.5 to 2.0 percent. In their opinion vermicompost should be used in bulk quantities

like other manures. The experiments of Jambhekar (1992) revealed that average C:N

ratio of earthworm treated waste was reduced to 15.9 : 1 where it was 31.1 : 1 in

untreated plot. The stimulation of biochemical activity and nutrient cycling by

earthworms increased the pasture productivity (Ross and Cairns, 1982).

Krishnamurthy et al., (1995) reported that the application of recommended dose of

fertilizers (RDF) along with vermicompost (2.5 t ha-1

) and Azospirillum (10 Kg ha -1

)

recorded the highest grain yield of Sorghum (5.08 t ha -1

).

Vermitechnology helps in increasing the production of crops. Kale (1991) has

attributed the improved growth in pastures and in other crops like rye and barley to

the chemical exudates of the worms and microbes in association with them. Tomati et

al., (1983) related the beneficial influence of worm cast to the biological factors like

gibberellins, cytokinins and auxins released due to metabolic activity of the microbes

harboured in the cast. The presence of compounds allied to Indole Acetic Acid (IAA)

in earthworm tissues has been mentioned by Kale (1993). It has also been indicated

that the chemical exudates of worms and those of microbes in the cast, influence the

rooting or shoots of layers. In a field trial Kale and Bano (1986) observed that the

seedling growth of rice in nursery increased significantly due to vermicompost

application, and transplanting of seedlings could be made one or two days earlier than

the usual practice. After transplanting the growth of seedlings in main field was more

favourably influenced by worm cast than the chemical fertilizer. This was attributed

to higher availability of nitrogen for plant growth. The improved growth was also

41

attributed to the release of plant growth promoting compounds from worm cast, which

in their opinion could easily replace the chemical fertilizers at nursery level.

The Nitrogen cycle:

Nitrogen is an essential component of all living organisms. A typical bacterial

cell for example contains about 12-15% nitrogen by dry weight, as components of

proteins, amino-sugars, nucleic acids and several other constituents of the cell. In

nature, the nitrogen occurs mainly in the lithosphere and as inert gas in the

atmosphere. Only a small part is found in the biosphere either in reduced forms as

ammonium-ammonia and amine groups or in many oxidized forms from nitric oxide

to nitrates, covering a wide range of oxidation states. Reduction-oxidation reactions

between the different stages of oxidation offer the potential of energy-generation by

microorganisms. A schematic representation of the biogeochemical cycle of nitrogen

is given in Fig 2.

Fig 2: Schematic representation of the nitrogen cycle

42

The gaseous nitrogen is fixed from the atmosphere into reduced nitrogen

compounds by free-living and symbiotic microorganisms, and then assimilated into

organic forms. In oxic environments, ammonia is converted into nitrite and nitrate

during the nitrification process, for which mainly two different groups of

microorganisms are responsible, the ammonia oxidizing and the nitrite-oxidizing

bacteria. The nitrate is further assimilated into organic material or reduced to nitrogen

oxides by denitrifying bacteria or completely into ammonia by the process of nitrate

ammonification, mainly operated by fermentative microorganisms. In anoxic

environments, ammonia and nitrite are converted into nitrogen gas by anaerobic

ammonia oxidation (Anammox), of which microorganisms of the order

Planctomycetales are responsible (Schmid et al., 2003; Strous and Jetten, 2004).

Our understanding of the nitrogen cycle is however far from complete, for

example with respect to the microorganisms that are involved; new processes and

players in the cycle evolve and are just beginning to be investigated and understood.

Moreover, over the past hundred years, human activity has dramatically altered the

global nitrogen cycle in several ways, for instance by increasing inputs of inorganic

and organic nitrogen through severe fertilization, by releasing nitrogen oxides in the

atmosphere by industrial combustion of fossil fuels, by acidifying soils, streams and

lakes, etc. Consequentially, human activities are altering the tendency of the processes

of the nitrogen cycle to balance each other in natural ecosystems. This suggests that

important knowledge on the nitrogen cycle is still missing and, even more, that

classical knowledge may need a reassessment, finally leading to a better

understanding of the overall nitrogen cycle (Jay Shankar Singh and Ajai Kumar

Kashyap, 2007).

43

The nitrification process

Nitrification is defined as the conversion of the most reduced form of nitrogen

(i.e. NH3) to its most oxidized form (i.e. NO3-). Soon after the identification of

nitrification as a biological process, Sergei Winogradsky succeeded in 1891 in

isolating nitrifying bacteria. He also confirmed the pioneer observation of Warington

that two different groups of bacteria cooperated in the process of nitrification, namely

the ammonia and the nitrite-oxidizing bacteria. Their isolation reveale their aerobic

nature and obligate chemolitho-autotrophic metabolism, except for the nitrite oxidizer

genus Nitrobacter which showed also chemo-organotrophic growth (Taylor and

Bottomley, 2006). The ammonia-oxidizing bacteria convert ammonia into nitrite

(equation 1), which is subsequently oxidized into nitrate by the nitrite-oxidizing

bacteria (equation 2).

NH3 + 1.5 O2 HNO2 + H2O (Δ G0‟ = -235 KJ mol-1

) (1)

NO2- + 0.5 O2 NO3

- (Δ G0‟ = -54 KJ mol

-1) (2)

At present, bacteria able to oxidize ammonia directly into nitrate have not

been described, although Costa et al., 2006, have recently postulated the existence of

bacteria able to carry out this hypothetical process, i.e., complete oxidation of

ammonia. Nitrification is also known to be performed by several heterotrophic

bacteria and fungi, able to convert either organic or inorganic reduced nitrogen forms

into more oxidized states (Brierley et al., 2001; Duggin, 1991; Jordan et al., 2005,

Killham, 1990; Prosser, 1989; Papen and von Berg, 1998). However, in heterotrophic

microorganisms the process is not likely to be generating energy (Burns and

Murdoch, 2005; De Boer et al., 1992; Laverman et al., 2000; Ross et al., 2004).

44

Nitrification in soil:

During the nitrogen mineralization process the termination of the reactions

occur at the point where ammonium is formed. This is the most reduced form of

nitrogen and serves as starting point for the process known as nitrification, the

biological formation of nitrite or nitrate forms contained reduced nitrogen (Arp et al.,

2002). In certain conditions, two separate and distinct steps are distinguishable in

nitrification. Since nitrate frequently appear during ammonium oxidation, it seemed

apparent to early microbiologists that the transformation involves an initial oxidation

to nitrite and later to nitrate. The conclusion has been made after the identification of

two separate groups of bacteria, each catalysing a separate reaction. Because nitrite is

rarely found in nature even in habitats where nitrification is proceeding rapidly the

nitrate formers must generally occur in the same environment as the ammonium

oxidizers.

Nitrification is an indicator of good soil biological activity and fertility.

Mineralization of organic nitrogen releases ammonia which may be subsequently

oxidized to nitrite by autotrophic bacteria like Nitrosomonas sps., Nitrosolobus sps.,

and Nitrosospira sps. and then to nitrite by other genera such as Nitrobacter sps., all

belonging to the family Nitrobacteriaceae (Patra et al., 2000). The resulting nitrate is

the major nitrogen source assimilated by plants. Besides autotrophic nitrifiers the

biological activity of nitrification is mediated by a diverse group of heterotrophic

microorganisms including several bacteria (Arthrobacter sps., Azotobacter sps.,

Pseudomonas flourescens., Aerobacter aerogenes., Bacillus megaterium) and Fungi

(Aspergillus flavus, Neurospora crassa, Penicillium sps.) [Falih and Wainwright,

1995; Mevel et al.,1996). However, heterotrophic nitrification is carried out at a

45

slower rate than autotrophic nitrification (Verstraete and Alexander, 1972; Watson et

al., 1981).

The first studies on the nitrification were conducted by Lees and Quastel

(1946, 1946a, 1946b). Using the soil perfusion apparatus, they confirmed it as a

comparatively slow process accomplished entirely by microorganisms. Further

experiments led to the conclusion that the rate of nitrification of a given quantity of

ammonium sulphate is a function of the degree to which the ammonium ions are

combined in or adsorbed on the soil in the form of the soils base – exchange

complexes. The greater the amount of adsorption, the faster is the nitrification. The

interpretation of these results was that the nitrifying bacteria grow on the surfaces of

the soil crumbs at the sites where ammonium ions help in base exchange

combinations and proliferate at the expense of such adsorbed ammonium ions.

In spite of various reports of the nitrifying ability of heterotrophic organisms

including fungi, there is a little doubt that the nitrification in soils is predominantly

effected by the autotrophic nitrifying bacteria. Provided that normal conditions for

microbial activities obtain that is, an adequate moisture, suitable aeration conditions,

favourable pH and source of nutrients, the autotrophic nitrifying bacteria multiply and

nitrify where ever ammonia and nitrite are available. In soils, nitrifiers are typically

present in low numbers ( 100- 1000/g) because they consume relatively large amounts

of NH4 or NO2 and produce a little biomass. Further more, nitrifiers must compete

with plants and heterotrphic bacteria for NH4. So their growth is further limited by

nitrogen availability. Nitrifiers are sensitive to pH. At high pH there may be problems

with NH3 while at low pH the formation of acid (HNO3) may be inhibitory. The

practical significance is that the process may be slowed, but not totally inhibited in

46

acidic soils (pH < 5). Numbers of nitrifiers in most soils are never very high, usually

some thousands per gram in a fertile arable soil, probably because of their slow

growth rate (Morill and Dawson, 1962) and their requirement for considerable

amounts of ammonia or nitrite as energy sources (Soriano and Walker, 1973). Given

favourable conditions numbers are largely dependent on the amount of ammonia

present and so numbers in sewage beds or in activated sludge may be much higher

than in soils. Nitrates are easily leached from soil where as ammonia is better

adsorbed and retained on the surface of the clay particles. Consequently, attempts

have been made to inhibit nitrification and so prevent losses of fertilizer nitrogen from

soil. Goring (1962) reported the use of the nitrification inhibitor N – serve [ 2-chloro-

6-(trichloromethyl) – pyridine] as a means of controlling nitrification in soil. Nitrogen

transformations occurring with in the biosphere are regulated most completely by

terrestrial and aquatic microorganisms (Todd et al., 1975).

Nitrification in Rhizosphere:

A great variety of abiotic and biotic factors shape soil and plant associated

habitats and modify the composition and activities of their microbial communities,

which in turn bear up on the quality of their environment, the growth of the plants and

the production of the root exudates (Bever et al., 1997). Bacterial communities in root

associated habitats respond with respective density, composition and activity to the

abundance and great diversity of organic exudates, eventually yielding plant species-

specific microflora which may also vary with plant development stages (Mahaffe and

Kloepper, 1997).

47

Root exudates selectively influence the growth of bacteria and fungi that

colonize the rhizosphere by altering the chemistry of soil in the vicinity of the plant

roots and by serving as selective growth substances for soil microorganisms.

Microorganisms in turn influence the composition and quantity of various root

exudate components through their effects on root cell leakage, cell metabolism and

plant nutrition. Based on differences in root exudation and rhizodeposition in different

root zones, rhizosphere microbial communities can vary in structure and species

composition in different root locations or in relation to soil type, plant species,

nutritional status, age, stress, disease and other environmental factors (Lupwayi et al.,

1998). Rhizosphere soil harbours higher populations of nitrifiers compared to non-

rhizosphere soils (Molina and Rovira, 1964).

Most nitrification is carried out by chemolithoautotrophic bacteria belonging

to Nitrobacteriaceae family. The family consists of two main groups, the ammonia

oxidizing or Nitrosobacter and the nitrite-oxidizing or Nitrobacter (Ramakrishna and

Sethunathan, 1982). Nitrifying bacteria are obligate aerobes and gain energy from the

oxidation of reduced nitrogen compounds to fix CO to organic carbon. Although

nitrite and nitrate are main nitrification products, there is increasing evidence that NO

and N2O are also produced as byproducts during autotrophic and heterotrophic

nitrification. Soil bacteria and fungi have the ability to oxidize reduced nitrogen

compounds (Simek, 2000).

Nitrification in forest ecosystems was reviewed by Smith et al., (1968).

Proposals for the importance of the nitrifying process on the functioning of forested

ecosystems have included an increase of nitrifying populations and nitrate losses from

watershed in New Hampshire. For many years, the process of nitrification was

48

thought to play a minimal role in N cycling in coniferous forest ecosystems. Based on

the physiological properties of a limited number of ammonia – oxidizing bacteria

(AOB) in pure culture, several soil factors were considered detrimental to them

including soil acidity, high C/N ratios, low N availability and the presence of

allelochemical compounds (De Boer et al.,1989).

Nitrifying bacteria:

Many microbiologists have investigated the abundance of these autotrophs in

nature. The numbers of ammonium oxidizers have been found to vary from zero up to

one million or more per gram of soil. The larger counts occur only in soils of pH

greater than about 6.0. In most habitats, species of Nitrosomonas and Nitrobacter are

found together. Otherwise nitrite might accumulate to phytotoxic levels. Populations

of both groups may be enlarged by use of ammonium salts and values in excess of 107

per gram are not unknown. In temperate climates, nitrifiers are numerous during the

warmth of the spring and rare during hot, dry summers and in winter, drying or

freezing decreases their abundance but never entirely eliminates these bacteria (Zeng

et al., 2003).

When ammonium is added to soil, autotrophic ammonium oxidizers and

subsequently, autotrophic nitrite oxidizers start to grow, with growth stopping when

all of the ammonium is oxidized to nitrate. Alexander and Clark (1965) estimated that

three times as a many ammonium oxidizers, such as, Nitrosomonas europaea, should

be produced during this nitrification process as nitrite oxidizers such as Nitrobacter

winogradsky. However, it is observed that nitrite oxidizers are numerically more

abundant in natural soils (Morill and Dawson, 1967; Volz et al., 1975, Lucas et al.,

2005).

49

Ammonification is the primary in situ source such as ammonium in

nonfertilized soils. External ammonium inputs include point sources such as sewage

or comparable waste substance amended to soil, as well as non-point sources such as

nitrogeneous fertilizers leaching from or applied to cropped lands. Continuous

enrichments of nitrifiers may occur downstream from a sewage plant or intermittent

population stimulation may occur from irregular flow from cropped soils due to

variation in rainfall patterns. Any nitrite found in soil is presumed to araise from

ammonium oxidation. Under normal situations, nitrite does not accumulate in soils

(Taylor and Bottomley, 2006). Thus, the limiting factor in nitrite oxidation to nitrate

in most soils is the conversion of ammonium to nitrite low levels of ammonium are

detected in most soils. The nitrite is usually converted to nitrate as rapidly as it is

formed. Under alkaline conditions, nitrite may accumulate. This results from the fact

that ammonia is toxic to Nitrobacter sps. Equilibrium between ammonium and

ammonia normally exists in soils with ammonium predominating in neutral and acidic

conditions and ammonia predominating under alkaline conditions. Thus, under

alkaline conditions, Nitrobacter sps. are inhibited but the ammonium oxidizers remain

active, hence nitrite accumulates. This phenomenon also occurs in microsites when

anhydrous ammonium is added to soil (Tate, 2000).

Virtually nothing is known of the genera that comprise a given nitrifying

population. The usual approach to the microbiological study on nitrification is that of

the Most-Probable-Number (MPN) technique used to obtain a statistical estimate of

the number of cells involved in a particular oxidative step (Papen and von Berg,

1998). The extent to which a given MPN medium may be selective in allowing the

growth of only a portion of the nitrifier population is an important consideration that

has not been studied. Members of a diverse nitrifier population probably contribute to

50

the nitrification process at quite diverse rates depending on individual growth rates

and niche circumstances (De Boer, 2001). If nitrification is to be understood and

predicted, it becomes necessary to gain a better understanding of diversity in the

nitrifying population and its implications with respect to activity in the environment

(Belser and Schmidt, 1978). An assessment of significance of nitrification in a

particular environment requires an understanding of the population ecology of

nitrifying organisms, a subject that has received scant attention (Belser, 1979).

Nitrifying bacteria grow slowly. Large quantities of ammonium nitrogen can

be converted to nitrate in soil. Broadbent et al., (1958) showed that between 7 and 88

lb of nitrate nitrogen per acre per day could be formed in loam and clay soils treated

with various ammonium fertilizers (100-16,00 lb N/acre). Morill and Dawson (1962)

found that the generation time of Nitrosomonas was half that of Nitrobacter at pH

values below 7.2 in soils perfused with ammonium and nitrite nitrogen respectively.

This means that nitrite was removed as soon as it was formed, a fact of some

importance since nitrite is phytotoxic. Generation times were pH dependent and

varied from 100 hrs at pH 6.2 to 38 hrs at pH 7.6 for Nitrosomonas and from 58 hrs at

pH 6.2 to 21 hrs at pH 6.6 and above for Nitrobacter.

Nitrifying microorganisms participate in the destruction of NO2 sorbed by soil

by oxidizing the nitrite formed as a sorption product. The nitrite oxidation rate

increases and shifts away from linearity as soil moisture increases to field capacity. At

constant soil moisture levels, the rate of nitrite oxidation in samples pre-exposed to

NO2 is greater than in samples not pre-exposed, suggesting adaptation and / or growth

of a nitrifying population (Ghiorse and Alexander, 1978, Jay Shankar Singh and Ajai

Kumar Kashyap, 2007).

51

Ammonia oxidation

Ammonia - Oxidizing Bacteria (AOB) perform the rate-limiting step of

nitrification, a key ecosystem process that in part determines the fate of nitrogen in

ecosystems. However, little is known about the factors that determine soil AOB

diversity or composition especially in tropical systems (Carney et al., 2004). The

potential importance of ammonium-oxidizing bacterial diversity or composition to

nitrification rates is unknown. Earlier studies have noted coincident patterns between

ammonium – oxidizing bacterial composition and/or diversity and nitrification rates,

but few others have attempted to study the affect of control the environmental factors

such as soil moisture, O2 or substrate availability and pH on nitrification rates

(Webster et al., 2002).

Ammonia-oxidizing bacteria are chemolitho-autotrophs that use the oxidation

of ammonia to nitrite as energy source and carbon dioxide as the major carbon source,

fixed via the Calvin- Benson cycle. Two electrons are required for the activity of the

Ammonium Monooxygenase enzyme, which catalyses the oxidation of ammonia

(Treusch et al., 2005). The other two enter the electron transport chain with

concomitant generation of a proton-motive force. The low energy gained by the

oxidation of ammonia as well as the reversed electron flow for the generation of

reducing power for the carbon fixation explains the low specific growth rate of these

bacteria. A doubling time of 8 hours has been reported in batch culture for the well

studied ammonia-oxidizing bacterium, Nitrosomonas europaea, (Ali Nejidat et al.,

2006). Cultivation-based methods are therefore time-consuming and difficult when

applied for the detection of these slowly growing-bacteria.

52

Nitrite oxidation

There have been several studies on the metabolism, survival and growth of

nitrite oxidizers in pure cultures and on their nitrifying activity in various

environments (Keen and Prosser, 1988). Although the biological conversion of nitrite

to nitrate is a well known process catalysed by Nitrite oxidoreductase enzyme since

the studies are hampered by inadequate methods of detection and counting (Degrange

and Bardin, 1995). Nitrite oxidizing bacteria grew to high populations without the

accompanying growth of ammonium oxidizing bacteria when nitrate but no

ammonium was added to a field (Volz et al., 1975). They had concluded that there

were two possible sources for the nitrite required for nitrite oxidizer growth. Hence,

nitrite would be produced as a result of the oxidation of ammonium mineralized from

soil organic nitrogen, in addition, to the product of nitrate reduction coupled to

organic matter oxidation in anaerobic microenvironments, with the resulting nitrite

diffusing into aerobic environments where it would be oxidized by the nitrite

oxidizers. If ammonium oxidation was the major source, one would expect that

growth of ammonium oxidizers would be required for growth of nitrite oxidizers to

occur. Activity of Nitrobacter population was strongly dependent on the population of

Nitrosomonas, but not vice versa (Belser, 1977). There are two growth phases of

nitrite oxidizers in the field that should be noted. During the initial phase of growth,

when the populations are small, nitrite is produced at a rate faster than it can be

oxidized, hence, nitrite will accumulate allowing the population to grow exponentially

(Ali Nejidat et al., 2006). At some point during the growth period, the rate at which

nitrite is being produced is lower than its utilization and at this point the nitrite

concentration in the soil will start to decline. When the nitrite concentration is reduced

sufficiently, the growth rate will decrease due to substrate limitation (Belser, 1977).

53

Enumeration of Nitrifying Bacteria by Most Probable Number method:

The most probable number (MPN) method is an important technique for the

microbiologist in the enumeration of viable count in samples of food, water, soil and

natural products. The method has undergone some evolution (Colwell, 1979), but the

principle itself is essentially unchanged, since it was first developed. Some samples

do not lend themselves to viable bacterial enumeration by any other procedure. Hence

the MPN technique is convenient and necessary in some instances and its use will

continue. Many uses for MPN have been described in the literature in the field of

microbiology related to water quality and public health (Hoskins, 1933). MPN

methods have been treated statistically by a number of investigators (Halvorson and

Giegler, 1933; Moran, 1954; Taylor, 1962; De Man, 1975; Finney, 1978, Rowe et al.,

1977).

The most probable number method allows for the first time estimation of

populations of bacteria capable of heterotrophic nitrification. The method is based on

the demonstration on the presence/absence of the nitrite/nitrate produced by

heterotrophic nitrifying bacteria during growth in a complex medium (Papen et al.,

1998). The medium in each tube was examined periodically by removing a few drops

aseptically to a spot plate depression containing NO2- test reagent (Strickland and

Parsons, 1960). If NH4+ medium gave a strong reaction (Dark red) when compared

with the uninoculated control, the tube was scored as positive for NH4+

oxidizers. The

tubes containing NO2 medium were scored positive for NO2 oxidizers, if the test

indicated that this substrate had decreased in concentration or disappeared. Tubes

scored negative and those showing weak reactions were reincubated. Control NH4+

medium developed a light pink reaction to dark pink as the experiment progressed.

Control NO2 medium did not change perceptibly.

54

The MPN technique is the most commonly used counting method for

nitrifiers. There are two requirements to get high counting efficiency. First, media and

growth conditions must be used that allow all the nitrifiers present to grow to a

population large enough to be detected. Second, the cells present must be sufficiently

separated from any particulate matter so that each cell will be individually dispersed

and can be transferred efficiently during the dilution process. These conditions do not

necessarily hold for commonly used MPN techniques and consequently populations

are often underestimated. Mathulewich et al., (1975) found that with the media they

developed, maximum MPN counts for ammonium oxidizers took between 20 and 55

days (Median, 25 days) to develop, where as the incubation time required for

maximum nitrite oxidizer numbers was at least 100 days. These incubation times were

reported as longer than three weeks at 280C (Alexander and Clark, 1965). Although,

the most probable number (MPN) viable counting technique for nitrification bacteria

is one of the most commonly used method in ecological studies of nitrification, it is

possible that under some conditions the MPN method greatly under estimates

indigenous nitrifier population (Rowe et al., 1977; Belser and Mays, 1982).

Autotrophic nitrifying microorganisms in soil were enumerated using the most

probable number technique (Lang and Ellicott, 1997). Viable autotrophic nitrifying

bacteria in environmental materials are usually enumerated by indirect most probable

number technique. In marine environments, MPN procedures have been used for both

groups. Some qualitative studies had used incubations lasting up to 90 days. Ideally,

the incubation should last just long enough to count for all of the inoculum cells

capable of growth under the conditions provided. The density of nitrifying bacteria is

estimated. Usually after 3 or 4 weeks of incubation for the examination ( Hashimoto

and Hattori, 1987). It is the only way to count Nitrobacter populations in soil. The

55

MPN – Griess procedure is selective ( Degrange and Bardin, 1995). MPN method was

also developed allowing for the first time estimation of populations of bacteria

capable of heterotrophic nitrification (Papin and von Berg, 1998).

To understand the population biology of nitrifying bacteria, the nitrate

producing activity of these organisms and the uptake of nitrate by higher plants has

been studied. It is concluded that all the three methods used in enumerating the

nitrifying bacteria, that is the MPN method, the fluorescent antibody technique and

the potential nitrification rate have serious draw backs (Woldendort and Laanbroek,

1989). The modified MPN procedure is used for statistical estimation of the numbers

of chemoautotrophic nitrifiers present in the nitrifying environment. The accuracy and

sensitivity of the MPN for quantification of these bacteria was unknown because no

standards exists (Fliermans et al., 1974). To enumerate the nitrifying bacteria and to

separate them from other soil microorganisms, advantage is taken of their

chemoautotrophic properties. In the case of Nitrosomonas, dilutions of soil are

inoculated into inorganic medium containing ammonium. The positive test for nitrate

in the inoculated medium but not in the controls indicates the presence of

Nitrosomonas. A negative test for nitrite, however is not sufficient evidence to prove

that Nitrosomonas is not present. It is possible that nitrite oxidizing bacteria as well as

ammonium oxidizing bacteria were present in the inoculum, and that during

incubation Nitrobacter group converted nitrite to nitrate or all nitrate formed by

Nitrosomonas. Because nitrate in the medium must come from nitrite produced from

ammonia by Nitrosomonas, a positive etst for either nitrite or nitrate in the inoculated

tubes and a negative test in the uninoculated control indicated the presence of

Nitrosomonas (Chendrayan and Umamaheswari, 2004).

56

Evidence on the production of nitrate is not sufficient to state that a pure

culture of Nitrosomonas has been obtained. Additional evidence to indicate absence

of other organisms is needed. To obtain such evidence the medium containing organic

materials that will permit many other organisms to grow must be used. If no growth

develops the parent culture of Nitrosomonas is probably, but not certainly a pure

culture.

The MPN technique is one of the means of determining the potential activity

of a microbial population and has been used by Alexander (1961), Smith et al.,

(1968); Focth and Joseph (1973) and Todd et al., (1975).

Comparison of the media for the growth and activity of nitrifiers:

One of the most important features to be considered in the study of bacterial

ecology is the choice of a proper medium. The nitrifying autotrophs are all obligate in

their reliance upon inorganic materials for energy: the organic carbon not utilizable by

these bacteria but their oxidative capacity is limited solely to nitrogen compounds and

no energy is obtained from other inorganic substrates. The carbon for cell synthesis is

derived from CO2, carbonate or bicarbonates while the energy for the reduction of

CO2 is obtained by the oxidation of the inorganic nitrogen compounds such as

ammoniacal nitrogen and nitrite nitrogen, hence growth does not occur in

conventional laboratory media (Alexander, 1978).

A number of media have been developed and used for studying the nitrifying

bacteria (Goldberg and Gainey, 1955; Engel and Alexander, 1958a; Lewis and

Pramer, 1958; Alexander and Clark, 1965 and Kannan, 2003). To obtain maximum

metabolic activity, enriched media may be recommended. Studies on nutrition and

biochemistry of autotrophic nitrifiers have been complicated because of the use of

57

culture media containing large quantities of insoluble constituents. The most common

component is calcium carbonate, included in the medium in order to neutralize the

nitrous acid produced by oxidation of ammoniacal nitrogen. Eventually, it was

concluded that adsorption to particulate ingredients was necessary for active

multiplication of the bacteria to occur (Imshenetsky and Ruban, 1954; Lees, 1955).

The autotrophic nitrifiers have an absolute requirement for oxygen and the process is

sensitive to pH, water and temperature (Lang, 1990). Optimum pH of the medium for

the growth of nitrifiers is between 6.6 and 8.0 or higher and the optimum temperature

of the medium for the growth of nitrifiers is between 30 and 350C (Alexander, 1978).

In the medium for ammonia oxidizers, it was found that 3 g of (NH4)2SO4/ litre is not

inhibitory, and higher concentrations would probably may not effect the process

(Engel and Alexander, 1958b).

Nitrification:

Nitrification may be carried out by both autotrophic and heterotrophic

microorganisms. The pioneering work of Winogradsky (1890) had established that

nitrification is typically associated with the metabolism of certain chemoautotrophic

bacteria. Two groups were distinguished, one deriving its energy for cell synthesis by

the oxidation of ammonium, the other by the oxidation of nitrite.

Autotrophic nitrification

Nitrifying bacteria are classified as obligate chemolithotrophs. This simply

means that they must use inorganic salts as an energy source and generally can not

utilize organic materials. They must oxidize ammonia and nitrite for their energy

needs and fix inorganic carbon dioxide to fulfil their carbon requirements. They are

largely non motile and must colonize a surface (Gravel, Sand, Synthetic biomedia

etc.,) for optimum growth. They secrete a sticky slime matrix which they use to attach

58

themselves. Species of Nitrosomonas and Nitrobacter are Gram negative mostly rod

shaped microbes ranging between 0.6-4.0 µ in length. They are obligate aerobes and

can not multiply or convert ammonia or nitrites in the absence of oxygen. Nitrifying

bacteria have long generation time due to low energy yield from their oxidation

reactions (Zeng et al., 2003). Since little energy is produced from these reactions they

have evolved to become extremely efficient at converting ammonia and nitrite.

Scientific studies have shown that Nitrosomonas bacterium is so efficient that a single

cell can convert ammonia at a rate that would require upto 1 million heterotrophs to

accomplish. Most of their energy production (80%) is devoted to fixing CO2 via the

Galvin cycle and little energy remains for growth and reproduction. As a

consequence, they have a very slow reproductive rate. Nitrifying bacteria reproduce

by binary division under optimal conditions Nitrosomonas may double in every 7 hrs

and Nitrobacter for every 13 hrs. More realistically they will double in 15- 20 hrs.

This is extremely long time considering that the heterotrophic bacteria can double in

short time as 20 mins. In this time that it takes a single Nitrosomonas cell to double in

population, a single E.coli bacterium would have produced a population exceeding 35

trillion cells. None of the members of Nitrobacteriaceae is able to form spores. They

have a complex cytomembrane (Cell wall) that is surrounded by slime matrix. All

species have limited tolerance ranges and are individually sensitive to pH, dissolved

oxygen levels, salt, temperature and inhibitory chemicals. Unlike species of

heterotrophic bacteria, they can not survive any drying process with out killing the

organisms (Alexander, 1961; Azam and Farooq, 2003).

59

The microorganisms linked directly to nitrification in natural environments are

the Gram negative chemosynthetic autotrophic bacteria comprising the family

Nitrobacteriaceae. These bacteria relate to corresponding stages of nitrification

(oxidation of NH4 to NO2 and further oxidation of NO2 to NO3) (Winogradsky, 1890).

The important bacteria for soil nitrification are Nitrosomonas europaea, Nitrosospira

briensis, Nitrosococcus nitrosus, Nitrosolobus multiformis (Ammonium oxidizers)

and Nitrobacter winogradsky, Nitrobacter agilis (Nitrite oxidizers) (Watson, 1974). A

new species of ammonium oxidizer Nitrosovibrio tenuis (Harms, 1976) and a new

genus and species of Nitrosococcus mobilis were described (Koops, 1976). In addition

to Nitrosomonas, several ammonia oxidizing bacteria were shown to oxidize

ammonia to nitrite or nitrate in defined media (Hutton and Zobell, 1953).

Heterotrophic nitrification:

In addition to chemolithotrophic nitrifying bacteria many other heterotrophic

soil bacteria and fungi have the ability to oxidize reduced nitrogen compounds both

mineral and organic in the process called heterotrophic nitrification. Heterotrophy is

related to use of organic compounds as source of carbon for biomass synthesis.

Heterotrophic nitrification may prevail in microsites with unfavourable conditions for

autotrophic nitrifiers as in acidic forest soils. As in autotrophic nitrification, nitrogen

gases may also be produced during heterotrophic nitrification, although the

significance of the both autotrophic and heterotrophic sources of NO and N2O has

been clearly demonstrated (Simek, 2000). Many heterotrophic microorganisms covert

reduced forms of Nitrogen to Nitrite. Among these heterotrophs, the ability to oxidize

ammonium or amino nitrogen is particularly common (Eylar and Schmidt, 1959).

Nitrogen, amines, amides, N-Alkyl hydroxyl amines, oximes, hydroximic acids and

aromatic nitro compounds serve as substrates for nitrite formation by individual

60

microorganisms. The heterotrophs do not make use of the energy released in

oxidation for growth any often the more oxidized products do not appear until active

proliferation has ceased. Although a large number of soil microorganisms (27% of the

actinomycetes, 26% of the bacteria and 17% of the fungi) produced nitrite in peptone

glucose broth. None of the isolates formed more than 2 ppm of nitrite nitrogen (Eylar

and Schmidt, 1959). Out of 161 starins tested 48 strains of actinomycetes produced

nitrite in a medium containing ammonium as a sole nitrogen source but the yield of

nitrite nitrogen was small. Nitrite also may be generated from compound containing

nitrogen at a level of oxidation higher than that found in ammonium. Species of

Pseudomonas, Proteus and Microbacterium produce nitrite which had been grown in

presence of hydroxyl amine. The pyruvic, oxalacetic and phenyl pyruvic oximes were

rapidly oxidized to nitrite when applied to soil. Species of Achromobacter and isolates

from soil found to form nitrite when grown in a medium with pyruvic oxime as the

sole source of carbon and nitrogen. Nitrate is likewise released microbiologically

from aromatic nitro compounds. Nitrate appears to be formed by fewer heterotrophic

microorganisms that are capable of generating nitrite from compound containing

nitrogen in a reduced form. As reported earlier (Eylar and Schimdt, 1959) out of 1331

isolates tested 20 are capable of forming significant level of nitrate from amino

nitrogen. Nineteen of these isolated were fungi, 17 being identified as strains of

Aspergillus flavus, one as penicillium sps and another as a Cephalosporium sps. Only

a single bacterial isolate was capable of forming nitrate in the test medium. A strain of

Arthrobacter globiformis has also been shown to oxidize ammonium to nitrate with

traces of nitrite and hydroxylamine accumulating in the culture liquid (Gunner, 1963).

It was reported that Pseudomonas sps. isolated from the soil enrichments containing

3- (p chloropheny)-1-dimethylurea to form nitrite and nitrate when incubated with

61

urea or certain urea derivatives (Doxtader and Alexander, 1966). There is increasing

evidence that in many acidic soils heterotrophic nitrifying bacteria may be more

important for nitrification than autotrophic nitrifiers, since autotrophs either can not

be detected in, or isolated from these soils, or cell numbers are too low to count for

the nitrate production observed. Therefore, it was assumed that in these soils

heterotrophic nitrifiers may represent the main, if not the exclusive, organisms of

nitrate production (Johnsrud, 1978; Kreitinger et al., 1985; Lang and Jagnow, 1986).

However this assumption remains speculative, since methods were lacking to

demonstrate that microorganisms with the potential of heterotrophic nitrification are

present in these soils and to count the cell numbers. Heterotrophic nitrification is

defined as the oxidation of ammonium/ ammonia or of organically bound nitrogen

from the oxidation state 3 to Hydroxylamine, Nitrite and Nitrate by heterotrophic

bacteria under aerobic conditions. In earlier times bacteria were regarded as

heterotrophic nitrifiers if they were able to produce hydroxylamine and/ or nitrite

from an artificial compound like pyruvic oxime (Castignetti and Gunner, 1980).

Effect of Environmental Factors on Nitrification

Nitrification is much more sensitive to environmental conditions than most

other nitrogen transformations which are carried out by a more diversed group of

microorganisms. The rate of nitrification increases with soil temperatures upto about

350C (95

0 F) and at below 5

0C (40

0F) very little NO3 is formed (Milton Maada-

Gomoh Saidu, 2009). Soil pH is also important. Below a pH of 6.0, nitrification is

inhibited by acidity and the process virtually ceases at a pH of 4.5 – 5.0. Under

alkaline conditions, production of NO3 markedly enhanced. The optimum pH is

normally between 7 and 8 but NO3 may be found at a pH of 9.0 or even higher

(Mulvaney, 1994). The community structure of ammonia oxidizing bacteria in soil is

62

influenced by different selective factors such as pH, gravimetric water content and

treatment with fertilizers (Kowalchuk and Stephen, 2001). Avrahami et al., 2003 had

found that temperature effects the community structure of ammonia oxidizers only

after long incubation (> 16 weeks) but not after short incubation <4 weeks (Avrahami

et al., 2002). Community structure was in addition influenced by fertilizer treatment

indicating that ammonium was also a selective factor for different ammonia oxidizing

populations. It seems to be desirable to study the effect of temperature and available

fertilizer in most soils with different communities of ammonia oxidizers. The effect of

the environmental conditions on mineralization and nitrification in saline soils have

not yet been investigated. It was observed that naturally saline coastal soils,

nitrification occurs as long as there is sufficient variation (SaadEddin, 1983).

Effcet of pH:

Physical and chemical factors affect the rate of ammonium oxidation. This fact

is simply demonstrated as nitrification rates differ according to soil type in sterile

soils receiving the same inoculum. Chief among the ecological influences is acidity.

Low pH led to decrease in number of nitrifiers (Paavolainen, 2000). Several careful

investigations have demonstrated a significant correlation between nitrate production

and pH. In acidic environments nitrification proceeds slowly even in the presence of

an adequate supply of substrate and the responsible species are rare or totally absent at

great acidities. Some soils nitrify at pH 4.5, while others do not (Leininger et al.,

2006). The difference is possibly attributable to acid adopted strains or to chemical

differences in the two habitats. The acidity affects not only the transformation itself

but also the microbial numbers. Neutral to alkaline soils have large populations and

the pH values for the growth of these bacteria depends to some extent upon the

locality from which they originated. Strains derived from acid soils are more tolerant

63

to high nitrogen ion concentration than those from areas of alkaline pH, certain

isolates of the former category having an optimum near 6.5 while 7.8 is preferred by

strains of the later group. The autotrophic bacteria, responsible for the oxidation of the

ammonium and nitrite (Nitrosomonas and Nitrobacter respectively), do not seem to

be affected by the initial pH value of the medium (Kiese et al., 2002). When

autotrophs can grow, the pH value of the medium is about 8.5, because it has been

buffered through out the organic matter mineralization by the heterotrophic

microorganism. This pH value is optimum for nitrifying bacterial growth which takes

place at higher rate (Owen et al., 2003). At the initial pH value of 12 there is a delay

of about 10 days before autotrophic microorganisms begun nitrifying. In this case, the

pH is buffered to about 9.0 and the nitrifying bacteria consequently need a period of

adjustment to the medium. The initial pH value of the medium thus influence the

maximum nitrite and nitrate concentrations. A higher initial pH value involves the

greater nitrifying bacterial selection. It was observed that the maximum

concentrations for nitrite and nitrate did not vary markedly when initial pH value is in

the range 8.5 – 11.0 (Avarez-Mateos et al., 2000).

With rare exceptions, sluggish nitrifying activity was evidenced when the soil

pH was below 5.5 and the responsible chemoautotrophs were found to be present in

very small numbers (measured by the MPN method). Yet the same soils usually

showed rapid rates of nitrification after they were adjusted to pH 7.0 or above (Morill

and Dawson, 1962). The nitrifiers can only carry out efficient nitrification with in a

pH range of 7.5 – 8.5. Out side of this range, the rate of nitrification slows to

generally unacceptable levels. For each pound of ammonia nitrified to nitrate, 7.2

pounds of alkalinity (as calcium carbonate) is destroyed. Because of the destruction of

alkalinity through the release of Hydrogen ions, sustained nitrification causes a drop

64

in pH. Sins and Collins (1975) reported that nitrification rates in soil markedly below

a pH of 6.0 and consequently, higher counts between 7.4 and 8.2, well with in the

neutral to alkaline range. Therefore as expected, little effort on the nitrification rate

could be observed. Optimum pH values may vary from 6.6 – 8.0. Typically,

nitrification rates in agricultural soils decrease below pH 6.0 and become negligible

below 4.5. High pH values inhibit the transformation of nitrite to nitrate (Paul and

Clark, 1996).

Effect of Temperature:

Temperature is an important factor which markedly affects certain

biochemical activities in different soils. It was shown that rate of nitrification

increases with increasing temperature from 8 – 280C (Focht and Verstreate 1977).

Mahendrappa et al., (1966) suggested that the environment exerts a more drastic

effect on Nitrobacter than on Nitrosomonas as also reported by Russell et al., (2002);

Roberta et al., (2006). There was no relationship between rhizosphere, surface or

atmospheric temperature and the activity of either ammonium oxidizers or nitrite

oxidizers around plant roots (Mahasneh et al., 1984, Kim et al., 2006). Nitrifying

activity of bacteria and concentration depends on specific free ammonia concentration

(ratio NH3/ biomass), that is a function of temperature, pH, ammonium concentration

and nitrifying biomass concentration. So, temperature is a key parameter in the

nitrification process producing two opposite effects: Bacterial activation and free

ammonia inhibition (Polanco et al., 1994). Temperature is one of the important factor

that influence nitrifier populations as reported by Belser (1979) and Mahendrappa et

al., (1966) who had reported that indigenous nitrifiers had temperature optimum in

correspondence to their climate region.

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Nitrifiers prefer moderate temperature ranging from 20-300C as temperature

decline will slower their metabolic activity. At temperature levels above 350C,

however, they enter a region of potential life threatening stress perhaps due to enzyme

destruction (Arunachalam and Arunachalam, 1999). Nitrifiers specifically have an

upper threshold of about 400C, at which point their activity completely stops, while

the permissible upper threshold for nitrifiers appears to be approximately 50C higher.

In either case, attention should be given to stabilizing reactor temperature relative to

avoiding extremes and short term transients (Alleman, 1984). Slow nitrification

occurs below 50C under snow cover in many soils. It is also slow below 40

0C. The

optimum is between 30-350C. The interaction of temperature, moisture, aeration and

other factors makes up the seasonal effects. In temperate areas, the nitrification

process is greatest in spring and fall slowest in summer (Underhill, 1990; Tate, 2000).

Inhibition of nitrification:

In soils, organic nitrogen is converted to ammonium through microbial

decomposition. Ammonium formed in soil, added as fertilizers, or in precipitation is

rapidly oxidized to nitrate in the nitrification process carried out by specific bacteria.

Nitrification results in the production of nitrate, a form of plant – available nitrogen

which is readily lost from soils. Nitrification inhibitors are chemicals that slow down

or delay the nitrification process thereby decreasing the possibility that large losses of

nitrate will occur before the fertilizers nitrogen is taken up by plants (Banerjee et al.,

1999). As revealed from researched reports from India, acetone extract of neem seed

crush could be used as nitrification inhibitors (Brains et al., 1971). Alcohol extract of

neem effectively conserved the ammoniacal nitrogen. Lipid associates of neem check

the conversion of ammonium to nitrite and the subsequent nitrate (Sahrawat and

Parmar, 1975).

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The majority of the research indicates that nitrification inhibitors, when

applied to soils in conjunction with N fertilizers or animal wastes, have benefical

effect on reducing nitrate leaching, nitrous oxide emission and, as a result increase

plant growth through increased N use efficiency (Merino et al., 2001). However, this

is not always the case as there are reports of variable or nil effects of nitrification

inhibitors on N losses and plant yields (Merino et al., 2001). A number of studies

throughout the United States have demonstrated that nitrification inhibitors effectively

retard the conversion of ammonium to nitrate in a variety of soils. Results indicate

that application of nitrification inhibitors delay the conversion of ammonium to nitrate

for 4-10 weeks depending upon soil pH and temperature with the fall in the

application of N fertilizers. Nitrification inhibitors minimize nitrification until low

soil temperatures (400F) to stop the process. The underlying concept in using

nitrification inhibitors is to increase the availability of NO3 and hence its vulnerability

to escape mechanisms as the later is directly proportional to the former. A great deal

of work has been done and reported on wastes to retard/ inhibit the rate of nitrification

not only to reduce fertilizer N losses (Aulakh et al., 1984) but also to prolong the

persistence of fertilizer nitrogen in ammonical form (Crawford and Chalk, 1993).

In recent years a number of chemicals including pesticides have been studied

to inhibit nitrification (Bremner and Bunday, 1974; McCarty and Bremner, 1989;

Feng and Barker, 1990; Lodhi et al., 1996). Some natural products like neem

(Azadiractica indica) cake are reported to inhibit the activity of nitrifiers (Sahrawat et

al., 1974).

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Impact of Pesticides on nitrification

Rapid increase in human population density and advancement in agricultural

technology has led to a greater release of xenobiotic compounds into the ecosystem

resulting in environmental pollution. The major sources of xenobiotics applied are

pesticides, which are in high demand these days to control major insect pests and

disease of important crops as well as to protect the agricultural products from

microbial spoilage during storage and transition. Recent incidences had shown the

presence of these toxic chemicals in drinking water (Mathur et al., 2003), and as well

terrestrial system that composed of soil (Singh et al., 2003a,b). Among the soil

biological processes, nitrification is much sensitive transformation of the major

elements (Subba Rao, 1999). Thus, nitrification is more sensitive to pesticide

treatment because nitrifying bacteria involved in the process are severely affected

(Graebing et al., 2002).

The present day cultivation technology requires a high input of synthetic

fertilizers besides using a load of insecticides, fungicides and herbicides in the field

(French and Gay, 1963). The entry of these agrochemicals into soil has far reaching

consequences as it disturbs the delicate equilibrium between microorganism and its

environment (Megharaj et al., 1986). A disturbance of this system by toxicants can

have profound influence on soil microflora and consequently soil fertility (Moorman,

1989; Martinez-Toledo et al., 1992). However, no definite conclusion can be made on

the effect of different insecticides on the growth and activities of microorganisms in

soil, since different groups of insecticides exhibit manifold variations in toxicity

(Matsumura and Boush, 1971).

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On the other hand many reports showed reduction in nitrification process in

fortified soils. Inhibitory effects of lindane and granular baygon on nitrification were

reported by Vertstraete and Vlassak (1973) and Kuseske et al., (1974), respectively.

Sahrawat and Parmar (1974) reported that azadirachtin, a general biological

insecticide, acts as a nitrification inhibitor. The insecticides malathion and parathion

at 10 and 50 µg/g soil rates considerably retarded the ammonium oxidation (Sahrawat,

1979). At 100 and 1000 ppm Cartap HCl reduced nitrite nitrogen and nitrate nitrogen

in soil (Endo et al., 1982). Fluchloralin at 5 and 100 ppm inhibited nitrification

(Palaniappan and Balasubramanyan, 1986). Thimet, bavistin and diuron at 10 and 100

ppm retarded the process of nitrification, and the observed inhibition increased with

increasing pesticide concentration (Sarawad, 1987). Nitrification was found to be

significantly inhibited in all the soil samples amended with aldicard or aldicarbsulfone

(Gupta et al., 1990). Likewise, fenoxyprop and tridiphame markedly retarded

nitrification at 50 mg/Kg in soil for three weeks. Kennedy et al., (1999) had reported

that the ammonium oxidation was depleted at higher levels of 1.0 and 1.5 Kg/ha of

carbofuran than normal field dose of 0.5 Kg/ha. Jana et al., (1998) reported a decrease

in nitrate accumulation with increasing dose of endosulfan. Similarly, Saxena and Rai

(1999) reported a substantial decrease of nitrification in soils on endosulfan

application. This may be accounted for the reduction in nitrifying bacterial population

as a consequence of endosulfan application. Bensulfuron and Cinosulfurol at normal

field application rates decreased the biological activity of nitrification (Gigliotti and

Allievi, 2001).

Fungicides are often potent nitrification inhibitors (Parr, 1974; Wainwright,

1977). Captan had inhibited nitrification upto 2 – 3 weeks as observed by Agnihotri

(1971). Similarly, low dosages of Dexon seem to have no pronounced effect on nitrate

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formation, but as the concentration of fungicide increased, nitrification was inhibited

(Aghnihotri, 1973). Mancozeb at 10 mg/ Kg soil decreased nitrification for 3 months

(Doneche et al., 1983). Kaushik et al., (1987) reported that fungicide Zineb inhibited

nitrification in soil at 5µg/g soil and above. Similarly, inhibition of nitrification was

reported with high concentrations of fungicide, ridomyl or dihydrel (Finkelstein and

Golovleva, 1988). Mancozeb, a contact fungicide had a direct effect on the population

of nitrogen fixing bacteria in rhizosphere and phylloplane and reduced the same

immediately after spray (Sudhakar et al., 2000).

Plant growth promotion:

In view of higher proportion of ammonifying organisms found among

rhizosphere isolates than among soil isolates, it is not surprising to find a more rapid

break down of amino acids in rhizosphere soil (Katznelson, 1960). While rhizosphere

provide a suitable environment for heterotrophic organisms, it may be less favourable

for autotrophic nitrifying bacteria. The effect of plant or plant communities on

nitrification changes with plant, plant age and the soil environment. Plants of different

age exerts different effects on the nitrifiers such as an increase of nitrifier population

at tillering and flowering stages of wheat (Riviere, 1960).