The nitrogen contents of pond soil normally varied between 0.08 and 0.6 per cent .

1. Role of Nitrogen Fertilizers in Fish CultureNitrogen is a very important component of many compounds including enzymes and chlorophyll, and essential for growth processes of phytoplankton. • Phytoplankton is essential for pond productivity which is related to fish growth and production. • Phytoplankton responds rapidly to applications of nitrogen. • This element encourages the growth of algae and aquatic macrophytes over pond surfaces and form a green color to the water. • Deficiency of nitrogen is evident when the pond water is clear and transparent.

2. When too much nitrogen fertilizers are applied, excess growth of algae occurs, water becomes more toxic, and upset the overall productivity of water. • Fish growth and reproduction is delayed or suppressed, and fishes are more susceptible to diseases, • Detrimental effects on fish food organisms should not result unless excessive quantities of nitrogen are used. • Although nitrogen is widely used for agricultural and aquacultural productivities, its potential adverse effects on water and soil qualities must be worth remembering.

3. Source and Distribution of NitrogenAlthough run-off water, atmosphere, plant debris, and dead animals are considered as the strategy of nitrogen supply to any water bodies, application of nitrogen fertilizers is a significant contribution to nitrogen input. • The elemental nitrogen in pond water is derived mostly from the air, the other source being bacterial denitrification of nitrate and ammonia. • Different species of blue- green algae such as Anabaena flosaquae, Microsystis aeroginosa, Nodularia spurn igelia etc. secrete extra cellular nitrogenous compounds.

4. The nitrogen contents of pond soil normally varied between 0.08 and 0.6 per cent. • One hectare of such a soil would likely contain about 3.6 metric tonnes of nitrogen while one hectare of atmosphere contains 300,000 metric tonnes of the element. • Therefore, the atmosphere is a limitless source of nitrogen although it is not readily usable by plants in elemental form. • Most of the pond soil nitrogen is in organic form. Nitrogen compounds are associated with clays and humus which protect them from microbial breakdown. • Ammonium ions are fixed by clay may account for up to 10 per cent of the nitrogen in surface soil and about 40 per cent in subsoils.

5. The quantity of nitrogen in the available ammonium and nitrate forms is seldom more than 2 per cent of the total soil nitrogen, except where nitrogen fertilizers have been applied. • Of course, these available forms are lost from soil through leaching and volatilization. Nitrogen Cycle The dominant nitrogen compounds of an aquatic ecosystem are nitrate-nitrogen, ammonia- nitrogen, dissolved molecular nitrogen, and large number of organic compounds from amino acids, amines to proteins and humic compounds of low nitrogen content.

6. Combined nitrogen occurs as hydroxyl amine, ammonia, nitrate, nitrite, and pariculate as well as dissolved organic nitrogen. • Organic nitrogen often accounts for more than one half of the total dissolved nitrogen. • Nitrogen undergoes a number of transformations that involve volatile, inorganic, and organic compounds. These transformations, however, occur simultaneously, but individual steps often accomplish opposite goals. The reaction may be viewed in terms of a cycle in which the nitrogen is shuttled back and forth at the discretion of microbial flora.

7. A typical nitrogen cycle of aquatic ecosystems consists of nitrogen fixation, ammonification, nitrification, and denitrification. • Different components of nitrogen cycle. terms of energy necessary for operation of the cycle are shown in Figure 1 • The steps from protein to nitrate provide energy for organisms that accomplish the breakdown, whereas the return steps require energy from sunlight or organic matter. • For example, ammonifying and nitrifying bacteria obtain energy from the breakdown, whereas, nitrogen fixing and denitrifying bacteria require energy from sunlight or organic matter.

9. ‘The term nitrogen cycle refers to the interaction among various forms of nitrogen in a pond soil, water, animals, and nitrogen in the atmosphere. • A schematic representation of the processes of nitrogen transformations and removal from the soil and water of a fish culture pond is given in Figure 2. • It has attracted scientific study for decades, and the practical significance of the cycle in a fish culture pond ecosystem for high production is beyond question

11. Fig. 2 : Nitrogen cycle in a fish culture ecosystem. it is a term for biogeochemical cycle involving the element nitrogen. Nitrogen is fixed by the blue-green algae and certain types of bacteria. The nitrifying bacteria convert total ammonia-nitrogen into nitrate and returns to the soil. • Under certain conditions, denitrifyirig bacteria obtain oxygen for respiration by breaking down nitrates and producing nitrogen gas to complete the cycle. • Organic manures, fertilizers, fish feed, faecal matter, and dead bodies of all organisms are the chief sources of nitrogen input in fish culture ecosystems. • Modern agriculture upsets the balance by applying chemical fertilizers to the soil, most of which is washed out into streams, rivers, lakes, and ponds.

12. The nitrogen is derived from chemical fertilizers, organic manures, fish feed, and dead plants as well as animals. • When total ammonia nitrogen is formed by the reduction of nitrogen, it is converted into nitrate and nitrite. These are consumed by algae and aquatic plants but after assimilation, enzymes help reduce nitrate to ammonia. Nitrogen depletion results from fish removal and absorption by plants. • Nitrogen fertilizers are readily soluble in water and soil solutions and are transformed into various forms (ammonia, nitrite arid nitrate) through micro-organisms. • Much of the nitrogen added to pond soil undergoes several transformations before it is removed.

13. The nitrogen in organic combination is first transformed to simple amino compounds (R - NH2), then to ammonium ion (NH4+) and finally to nitrate ion (NO3-). Even, nitrogen is either appropriated by micro-organisms or lost by denitrification and volatilization. Classification of Nitrogen CycleThough bulk quantities of nitrogen is present in soil as organic form, it is not largely available to plants. • The organic nitrogen pool is several times higher than that of inorganic one (Figure 17.3). However, scientific effort has been devoted to the study of organic nitrogen regarding its release to forms usable by plants. • The transformation of inorganic nitrogen to organic form of nitrogen is called immobilizationwhereas slow release of organic nitrogen to inorganic one is called mineralization

15. Mineralization and ImmobilizationThe release of organically bound nitrogen to inorganic forms (such as ammonium and nitrate) is called mineralization. • Soil organisms simplify and hydrolyze the organic nitrogen compounds and produces NH4 and NO3- ions. • The reverse process of mineralization such as conversion of inorganic nitrogen ions (NH4 and NO3-) into organic forms, is called immobilization. It occurs when animal and plant residues are added to pond soils. • Residues are degraded by micro-organisms which, in turn, absorb the inorganic nitrogen ions and convert to organic tissue where the nitrogen is immobilized.

16. After death of micro-organisms, some of the organic nitrogen in their bodies are converted into forms that make up the humus complex and some are released as ammonium and nitrate ions. • Fate of Ammonium CompoundsThe ammonium nitrogen is moved in the following directions: 1. Considerable amounts of ammonium nitrogen are appropriated by soil micro-organisms. Consequently, nitrogen is incorporated in their bodies.

17. 2. Phytoplanktons are about to use this form of nitrogen. 3. The ammonium ions are subject to binding by vermiculite (naturally-occurring mineral composed of shiny flakes, resembling mica) and organic matter. • In fixed forms, the nitrogen is not subject to oxidation, of course, in time it may become available. 4. Some ammonia gas is lost through volatilization particularly in alkaline soils. Such losses are significant when large quantities of ammonia are applied as fertilizers. 5. The remaining ammonium compounds are oxidized by bacteria, first to nitrites and then to nitrates. The entire process is called nitrification.

18. Fixation of AmmoniaIngeneral, both inorganic and organic soil nitrogen have the ability to fix or bind the ammonia. It is also important to note that different compounds and mechanisms are involved in these two types of fixation. Fixation by Organic MatterWhen ammonium-containing fertilizers are added to the pond soil, it reacts with soil organic matter and form compounds that resist decomposition. Therefore, it can be said that ammonia is chemically fixed or bound by the organic matter. Though it is not known the exact mechanisms by which ammonium fixation occurs, some specific reactions with components of humus have been established.

19. The reactions take place in the presence of oxygen and at high pH values. Since fixation capacity of ammonium is high in organic soils, the reactions result in a loss of available nitrogen that recommend the application of fertilizers. • Fixation by Clay MineralsSeveral clay minerals are able to fix ammonium ions. The ions are fit between the crystal units of clay minerals and consequently, fixed or trapped. • The ions are held in anon-exchangeable form, from which they are released to micro-organisms.

20. Nitrification It is a process of enzymatic oxidation of ammonia to nitrates by micro-organisms in the pond soil. The oxidation process takes place in two steps. In the first step, nitrite (N02) ions are produced by the bacteria Nitrosomonas sp., apparently followed immediately by further oxidation to the nitrate (NO3-) form by the bacteria Nitrobactor sp, First Step:NH4+ HONH2 ½ HONNOH Ammonium Hydroxylamine Hyponitrite N02 + H + Energy Nitrite

21. Second Step: NO2 N03 + Energy Of the two above-mentioned reactions, the second one is very significant because it prevents any great accumulation of the nitrite. • It is highly significant because nitrites are generally toxic to fish and other aquatic animals. Conditions Affecting Nitrification The nitrifying bacteria are more sensitive to their environment than most heterotrophic organisms. • Soil conditions greatly influence the activity and population of nitrifiers (autotrophs) and therefore, the efficiency of nitrification will receive brief consideration.

22. 1. Level of Ammonia: The process of nitrification occurs where there is a source of ammonia. High C/N ratio of residues prevent nitrification. Moreover, concentration of ammonia at high level also constraints nitrification. • Application to alkaline pond soils of urea or anhydrous ammonia, appear to be toxic to nitrobactor, resulting in accumulation of toxic levels of nitrite ions. 2. Temperature: The temperature favorable for nitrification is 25-35°C. • Therefore, tropical and sub-tropical fish ponds show rapid rate of nitrification and, consequently, the ability of ponds to provide nitrate for phytoplankton is enhanced. In temperate fish ponds, on the other hand, the accumulation of nitrate ions through nitrification is a bridged. • Nitrification rates gradually decline at temperature above 35°C and cease at temperature above 50°C.

23. 3. pH and Exchangeable Base-Forming Cations: • In acidic ponds, where pH value is below 6, there is remarkable accumulations of nitrate ions. Nitrifications rates decline at ponds where pH of soil is more than 7.5. • The rate of nitrification proceeds rapidly where the levels of exchangeable base-forming cations (such as Ca2 Mg, K, and Na) are high. The absence of these cations accounts in part for the slow nitrifications in acidic pond soils thus for the seeming sensitivity of the organisms to a low pH value. 4. Pesticides: Nitrifying bacteria are quite sensitive to some pesticides. Accumulation of pesticides to aquatic ecosystems through surface run-off almost completely inhibited the process of nitrification; of course, there are some pesticides which slow the process of the nitrification down  

24. . 5. FertilizersNitrifying organisms seem to have nutrient requirements. Therefore, application of nitrogen and/or phosphorus fertilizers is necessary to stimulate nitrification process. Use of large amounts of ammonium compounds to highly alkaline pond soils should be avoided to prevent loss of ammonia gas and to alleviate negative effects of the ammonium ion on nitrification process. Fate of Nitrate NitrogenThe nitrate nitrogen of the pond soil may go in three directions. It may • (1) be leached from the soil, • (2) be incorporated into micro-organisms, or • (3) escape from the pond as a gas.

25. Leaching and Gaseous Loss • Negatively charged nitrate ions are not adsorbed by the negatively charge soil colloids, they are subject to ready leaching from the soil and moved downward with the water. • It has been calculated that about 15 per cent of the nitrogen is lost by leaching. • If excessive fertilization is made in sandy and sandy-loam pond soils, nitrate loss is increased by leaching but such losses can further be accentuated by increased use of organic manure of compost. • Accumulation of nitrate in drainage water may rise to levels that could toxic to aquatic animals, and livestock. Therefore, the need to take necessary steps to minimize nitrate leaching is obvious.

26. In pond mud, the process of denitrfication is carried out by micro-organisms. This process involves chemical reactions and consequently gaseous losses of nitrogen take place. • Use of Nitrate-Nitrogen by Micro-organisms Both soil and plant micro-organisms readily assimilate nitrate-nitrogen. • If soil micro-organisms are provided with food, they use nitrates more rapidly than higher plants. • On the other hand, if supplemental fertilizer nitrogen is not applied, they will suffer from nitrate-nitrogen.

27. DenitrificationReduction by Micro-organismsBiochemical reduction of nitrate-nitrogen to gaseous form is a common phenomenon. Facultative anaerobic forms of micro-organisms take active part during the process of reduction. The five-valent nitrogen in nitrate is reduced stepwise to the zero-valent elemental nitrogen as noted below: 2NO3_ 2NO2_ 2NO N2O N2 (+5) (+3) (+2) (+1) (0)

28. Each step in the reaction is accelerated by a specific reductase enzyme. • It is important to note that the reaction can stop at any stage and the gaseous forms of NO, N20, and N can be released to the atmosphere. The oxygen atoms become incorporated into the bodies of the anaerobic bacteria.

29. Chemical Reduction In this process nitrite ions in a slightly acid soil will produce nitrogen gas when brought contact with urea, phenol, and carbohydrates. • The reaction is strictly chemical and does not require either the adverse soil conditions or the presence of micro-organisms and its practical significance is not great. The following reaction suggests what my happen to urea: CO (NH2)2 + 2HN02 CO2 + 3H20 + 2N2 UREA

30. Amount of Nitrogen Loss Through Denitrification In general, the exact magnitude of the losses of nitrogen through denitrification depends on the soil and cultural conditions. • Substantial losses of nitrogen through leaching and uptake by various species of plants and animals from different pond soil conditions, however, are not being uncommon. • In rice-fish cultivation, loss of nitrogen by denitrification may be very high. About 65 per cent of the applied fertilizer nitrogen is volatilized as elemental nitrogen. • By preventing the formation of nitrates by nitrification, losses can be reduced.

32. Ammonification*A number of anaerobic or aerobic heterotrophic protein mineralizing bacteria, fungi, and actinomycetes in water and soil utilize organic nitrogen-rich substrate and convert it to ammonia. This process is called ammonfi cation. The steps in ammonification are roughly the opposite of assimilation and amination. Process of AmmonificationThere are three ways of producing ammonia from organically bound nitrogen: • (1) extra-cellular organic nitrogen-containing compounds, biochemically or chemically, • (2) from living bacterial cells during endogenous respiration, and • (3) from death and lysed cells.

33. 1. Breakdown of Extra-cellular Compounds: • Complex nitrogen-containing organic compounds are first deaminated to ammonia by enzymes on the cell wall and then transports the ammonia to the cell where it is used for synthesis. • In organic wastes the amount of nitrogen-containing compounds is such that all the organic nitrogen is used in cell synthesis, but in cases where there is an excess of these compounds (low C/N ratio), ammonia will be formed in amount equal to the excess of the requirements for growth. • The keto-acids and hydroxy-acids of the compounds are used for energy purposes.

34. 2. Endogenous Metabolism • Although the process of endogenous metabolism is very complicated, it is known to take place in the presence of exogenous substrate (or endogenous substrate), so that even during growth there is the possibility of ammonia formation. • The metabolism depends on the composition of the cells and environmental conditions. • The rate of degradation of individual substances depends on their concentrations within the cells, which in turn depend on the nutritional status of the organism. • Amino acids, proteins and ribonucleic acids give rise to ammonia. In fact, the presence of glycogen prevents the breakdown of nitrogen-containing substances so that ammonia is not released until the “shortage products” have been used.

35. In cells which do not contain glycogen , an imbalance between the essential components of the cell sometimes occurs when there is an amino acid deficiency. Thus, excess ribonucleic acid formed is broken down. • 3. Death and Lysis: The cells which respire endogenously, lose their viability, that is, reproduction is hampered but continues to respire until lysis is reached when the cell membrane dissolves because of enzyme attack. Cell contents are exposed to the environment and production of ammonia occurs.

36. Proteolytic Activity of Micro-organisms Methylamines are produced by algae in freshwater and are likely to be decomposed by bacterial action to give ammonia. • Protein-mineralizing bacteria can contribute to the liberation of ammonia which thus becomes available to the algae as a source of nitrogen either directly or after oxidation to nitrate. • It has been suggested that the protein decomposition was initiated by proteolytic bacteria by hydrolyzing proteins, into peptides, and urea which in turn, are metabolized by ammonifying bacteria to liberate ammonia.

37. Organic nitrogen supplied by phytoplanktonic production or decomposition in water consists of about 85 per cent proteins and peptides. • These organic materials with high molecular weight cannot be directly taken up by bacteria. • Therefore, the hydrolysis of these materials is a first step of organic nitrogen utilization by micro-organisms

38. Ammonifying BacteriaA variety of micro-organisms such as Aerobactor cloacai, Pseudonions sp., Bacillus subtilis, Proteus vulgaris, and Escherichia coli are capable of hydrolyzing proteins into simpler compounds which in turn, are metabolized by ammonifying bacteria to liberate ammonia to ammonium sulfate. Ammonia is associated with a waste product overflow in microbial metabolism, the accumulated ammonium representing the amount of substrate nitrogen in excess of the microbial demands. 

39. Some of the ammonifying bacteria are substrate specific. They use only peptone but not amino acids, or use urea but not uric acid. • Some other ammonifying bacteria are able to use a wide variety of organic nitrogen sources. • Bacteria removes amino group and utilize the resulting ammonia as a source of nitrogen. Organic matter in the pond mud is transformed into ammonia by ammonifying bacteria.

40. Sources and Estimates of Ammonia in Fish Culture Ponds • It is known to all that ammonia play a key role in limiting fish growth in intensive fish culture ponds where fish production is dependent upon the application of protein-rich feed which serve as an important source of ammonia. • The diets which are used are often far from balanced and about 80 per cent of the feed nitrogen is excreted by the fish as ammonia. In addition, decomposition of unconsumed feed and the use of chemical fertilizer and organic manure further add ammonia to fish ponds. • Excretion of ammonium by zooplankton and autolysis after cell death are chief sources of ammonia in fish ponds.

41. The trapping and binding of ammonia in the pond sediments seems to be an another factor determining ammonia concentrations in the water. • The sediments serve both as source from which ammonia may diffuse into the pond water and a sink and trap for nitrogenous matter from the water. Ammonia Pool High concentrations of ammonia are found adsorbed on to the soil particles. The size of the ammonia pool in the growth cycle of an intensive fish pond in Israel has shown that the concentration of ammonia is increased along with the increase in sediment depth (Figure 17.4) while the size of the ammonia pool did not change during the growth cycle. Generally, ammonia pool is reduced when the ponds are dried.

43. Most of the intensively cultured fish ponds in Israel are characterized by a low ammonia content (below 0.1 mg NH3-N/l) for most of the growth period. • Experiments have, however, shown relatively high concentration of ammonia only early in the season but rapidly declined to trace amounts (Figure 17.5). • This clearly indicates effect of drying and refilling of the pond. • Immediately after refilling the ponds accumulation of ammonia in the sediments resumed, and in the water column a transient increase in ammonia (Figure 17.6) and nitrite was observed.

44. In some cases, this temporary increase in nitrite upon refilling could be related to fish mortality, since loss of nitrite from the water causes disappearance of nitrite toxicity. • At the time of drying the accumulated nitrate in the sediment is lost by denitrification upon refilling of the ponds. • In drained fish ponds, ammonia concentrations in the sediment progressively increased, reaching a peak at the end of the growth period. • Most of the ammonia is bound by soil particles, and only a small fraction remains free in the core water.

46. Ammonification in Relation to Fish Culture SystemThe rates of ammonification varied markedly among the fish culture system. • For example, a significant difference has been observed in the ammonification rate among three fish culture systems (such as traditional, mono and polyculture) with minimum activity in the traditional system and maximum in monoculture. • The maximum nitrogen input in the monoculture pond is responsible for the massive development of ammonifying bacteria. The rate of ammonification is, however, extremely variable in different seasons of the year and also in different fish culture systems. • Such changes are directly dependent upon the population dynamics of ammonifying bacteria.

48. Nitrogen Fixation • The fixation of nitrogen is the biochemical process by which elemental nitrogen is combined into organic forms. It is carried out by a number of organisms including a few actinomycetes, blue- green algae, and several species of bacteria. • The quantity of nitrogen fixed globally each year through different systems is enormous, having been estimated at about 170 million metric tonnes. • This exceeds the amount used in chemical fertilizers. Nitrogen fixation is said to be the most important biological process on earth.

49. System of Fixation • Biological nitrogen fixation occurs through a number of systems such as symbiotic fixation with legumes, symbiotic fixation with nodule-forming non-legumes, symbiotic nitrogen fixation without nodules, and non-symbiotic nitrogen fixation. • Of all the systems mentioned, the non- symbiotic nitrogen fixation is common in fish culture pond ecosystem. • Pond soil and water certain free-living micro-organisms that are able to fix nitrogen. • Since these organisms are not directly associated with plants, the transformation is called as non-symbiotic or free-living.

50. Fixation by AutotrophsThe light and the bacteria/algae constitute the system for this type of nitrogen fixation. • In the presence of light, certain photosynthetic bacteria and blue-green algae are able to fix both nitrogen and carbon dioxide simultaneously. • Though the contribution of the photosynthetic bacteria is not certain, blue-green algae is thought to be some significance. • Under normal conditions of an aquatic Freshwater Fish Culture ecosystem, about 25 mg N/hectare is fixed each year. • This estimate is no doubt to much variation depending upon the conditions peculiar to a particular ecosystem.