Industria, Comercio y Servicios

Most soil microorganisms rely on the inorganic soil nitrogen forms nitrate and ammonium as a nutrient supply. However, the bulk of the nitro- gen in most soil systems is contained in the organic fraction of the soil, and is not directly bioavailable. As soil organic material is oxidized by various soil fungi and bacteria, excess nitrogen may be converted to inorganic forms. If organic matter is sufficiently rich in nitrogen, degradation of the organic matter will result in the release of ammonium and nitrate nitrogen, or mineralization. On the other hand, degradation of organic matter with low levels of nitrogen will consume or immobilize ammonium and nitrate nitrogen as degrading microor- ganisms scavenge available nitrogen from the soil system (Stevenson and Cole 1999). In general, decomposition of organic materials with a C:N ratio of less that 35:1 will mineralize nitrogen, whereas degradation of those with a C:N ratio greater than 35:1 will immobilize nitrogen (Vigil and Kissel 1991).

Nitrogen mineralization can be divided into ammonification (Eq. (8.1)), con-

version of organic nitrogen to ammonia (NH3) which hydrolyzes in water to form

ammonium (NH4+) (Eq. (8.2)); and nitrification, which is the oxidation of ammo-

nium nitrogen to the nitrate form (NO3−). In ammonification, organic nitrogen

compounds such as proteins, amino sugars, and nucleic acids are biologically degraded into ammonium by a wide range of aerobic and anaerobic heterotrophs (Jansson and Persson 1982; El-Shinnawi et al. 1993). In nitrification, ammonium is oxidized to nitrate largely by autotrophic soil bacteria. In autotrophic nitrifica- tion, the conversion of ammonium takes place in two steps: the transformation

of ammonium into nitrite (NO2−) by oxidizing bacteria such as Nitrosomonas, and

the oxidation of nitrite into nitrate by Nitrobacter (Paul and Clark 1996; Watson et al. 1989). Oxidation of nitrite usually proceeds at a more rapid rate, so it is a relatively rare form of inorganic soil nitrogen (Stevenson and Cole 1999). Ammonium and nitrate make up the bulk of soil inorganic nitrogen, and are the principal forms available for bioremediation.

Ammonification:

R− NH2+ H2O→ NH3+ R − OH + energy (8.1)

Ammonia hydrolysis:

Table 8.1 Commonly used nitrogen fertilizers, nitrogen content, and chemical composition.

Fertilizer % Nitrogen Composition

Ammonium nitrate 33 NH4NO3

Ammonium sulphate 20.5 (NH4)2SO4

Calcium nitrate 16 Ca(NO3)2

Urea 45 (NH2)2CO

Anhydrous ammonia 82 NH3

Diammonium phosphate 20 (NH4)2HPO4

Ammonium polyphosphate 10–15 (NH4PO3)n

First step of nitrification:

2NH+₄ + 3O2→ 2NO−2 + 2H2O+ 4H+ (8.3)

Second step of nitrification:

2NO−₂ + O2→ 2NO−3 (8.4)

Nitrate is an anion and, as such, is repelled by negatively charged soil colloids. Nitrate salts are highly soluble, so nitrate moves with soil water and can easily be leached through soil material. Furthermore, nitrate can be reduced through the process of denitrification (Eq. (8.5)). Denitrification, reduction of nitrate to

nitrous oxide (N2O) or dinitrogen (N2) gas by heterotrophic anaerobic bacteria,

can thus be a major mechanism for nitrogen loss from poorly aerated soil sys- tems. Oxidation of organic substrates provides energy and carbon for denitrifying bacteria and nitrate acts as the terminal electron acceptor. Nitrate addition has been used to enhance anaerobic hydrocarbon degradation through this process, although the nitrate is used primarily as an electron acceptor rather than as a nutrient per se (Hutchins et al. 1991).

Denitrification: 2HNO3 +4H_→ −2H2O2HNO2 +2H_→ −2H2O2NO +2H_→ −H2ON2O(gas) +2H_→ −H2ON2(gas) (8.5)

Where supplemental nitrogen is required for bioremediation, there are many fertilizer materials that can be used. Some common fertilizers are listed in Table 8.1. Ammonium nitrate, ammonium sulfate, and calcium nitrate are com- monly used inorganic salts of nitrate or ammonium. All are highly soluble, and immediately provide nitrogen in a bioavailable form. There is some disagree- ment on the efficacy of various inorganic nitrogen salts for bioremediation.

Chang and Weaver (1997) noted a preference for ammonium versus nitrate by petroleum degrading soil bacteria. Brook et al. (2001) reported that ammonium sulfate was more effective than either ammonium nitrate or potassium nitrate. On the other hand, Rasiah et al. (1992) compared calcium nitrate, ammonium nitrate, sodium nitrate, ammonium chloride, and potassium nitrate, and found that adding nitrate salts to a soil contaminated with oil refinery waste increased biodegradation more than ammonium. They also reported that the counter-ion had an effect on degradation, with calcium nitrate being the most effective, and effectiveness decreasing as the counter-ion was changed in the following order: Ca > Na > K > NH4. Wrenn et al. (1994) compared ammonium chloride and potassium nitrate in solution culture and found that ammonium chloride was much less effective, and attributed the difference between the two sources to acidity produced during nitrification of the ammonium nitrogen (see Eq. (8.4)). When pH was controlled, there was no difference between the two sources.

Urea is a simple organic nitrogen salt. It is highly soluble, but must be hydrolyzed by the urease enzyme to form ammonium before it can be utilized (Eq. (8.6)). Frankenberger (1988) noted that soil urease activity is inhibited by petroleum materials, limiting the effectiveness of urea as a nitrogen source in bioremediation. On the other hand, both Brook et al. (1997) and Lee and Silva (1994) found that hydrocarbon degradation rates were higher in urea amended soils than in soil fertilized with ammonium nitrate. Brook et al. (2001) reported that urea was superior to potassium nitrate at a C:N ratio of 40:1 and to ammo- nium nitrate or potassium nitrate at a C:N ratio of 20:1. If urea is used, attention should be paid to soil pH. During hydrolysis, soil pH can rise dramatically, and it can subsequently drop below the original soil pH as ammonium is nitrified (see Eq. (8.3)).

Urea hydrolysis:

CO(NH2)2+ 3(H2O)→ 2NH3+ CO2+ 2H2O→ 2NH+4 + CO2+ 2OH− (8.6)

In addition to inorganic nitrogen salts, there are a number of slow-release or controlled-release nutrient sources. These materials may be coated fertilizers

(sulfur-coated urea and Osmocote

_®

are examples), slowly soluble materials (such

as metal ammonium phosphates), or materials that must be microbially mineral- ized to release nitrogen (organic fertilizers and urea formaldehydes, for example. Several studies have compared stimulation of bioremediation with controlled-

release versus soluble nutrients. Cunningham (1993) found that MaxBac

_®

, a

fertilizer coated with a selectively permeable membrane, resulted in more rapid diesel fuel degradation than a conventional nutrient source. In contrast, a urea oligomer was less effective when added to a diesel fuel contaminated soil than

either urea or ammonium sulfate (Brook et al. 2001). Croft et al. (1995) compared

MaxBac with Inipol EAP22

_®

(an oleophilic urea-based fertilizer), and reported

that MaxBac fertilized soil was only marginally better than unfertilized soil, whereas soils fertilized with Inipol EAP22 showed a significantly better response. Oil-based fertilizers such as Inipol EAP22 are thought to be advantageous at coastal-contaminated sites where loss of soluble nitrogen can be extreme.

To maximize effectiveness, controlled-release nutrients should be released at a rate equivalent to microbial demand (see Chapter 4, Section 4.2.2.2, and discussed in Chapter 11, Section 11.3.2.2). It has been suggested that organic nitrogen sources that must be microbially decomposed to release nitrogen might best match microbial nitrogen demand during hydrocarbon degradation (Walworth et al. 2003). Walworth et al. (2003) demonstrated the effectiveness of a fish processing by-product as a controlled-release nutrient source. Lee and Silva (1994) also successfully used a urea-fish-meal mixture for degrading Prudhoe Bay crude oil.

Ammonia, a gas at atmospheric pressure and ambient temperatures, is an effective nitrogen source which can be injected into soil. Ammonia is highly water soluble. As it hydrolyzes to form the ammonium ion, hydrogen ions are consumed and soil pH can be raised to 9 or higher (Tisdale et al. 1993), which can adversely affect soil microorganisms in close proximity to the fertilizer (see Eq. (8.2)). At pH values of 8.0 and above, volatilization of ammonia gas can cause large losses of nitrogen if it is not incorporated into the soil. As in the case with urea, the final pH of soil fertilized with ammonia may be lower than that of unfertilized soil because of acidification caused by nitrification of ammonium to nitrate (see Eq. (8.4)).

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