Chemistry: molecular biology and microbiology – Process of utilizing an enzyme or micro-organism to destroy... – Destruction of hazardous or toxic waste
Reexamination Certificate
2000-02-04
2003-04-22
Redding, David A. (Department: 1744)
Chemistry: molecular biology and microbiology
Process of utilizing an enzyme or micro-organism to destroy...
Destruction of hazardous or toxic waste
C435S262000, C210S610000, C210S747300
Reexamination Certificate
active
06551815
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention (Technical Field)
The present invention relates to methods and apparatuses for in situ denitrification, most particularly biodentrification.
2. Background Art
Bioremediation is the use of microorganisms to convert harmful chemical compounds to less harmful chemical compounds in order to effect remediation of a contaminated site. The microorganisms are generally bacteria but can be fungi. The contaminants can be organics such as petroleum hydrocarbons and domestic wastewater or inorganics such as nitrate and metal ions. Microbial growth and metabolism require suitable nutrients to construct new cells and materials to supply energy through oxidation-reduction reactions.
The nutrients that are used to construct new cell components include inorganic or organic compounds that provide the major elements (carbon, oxygen, hydrogen, nitrogen, sulfur, phosphorus, etc.) to the cell. Elements that are not major components of building block material but are still necessary for growth (micro-nutrients) include such elements as Mg, Ca, K, Fe, Mn, Na, Zn, and Cl. Groundwater contaminated with nitrate and sulfate requires addition of only phosphorus nutrient and hydrocarbon substrate for the supply of major elements. While a variety of minerals are required by microorganisms, they are needed in trace amounts, and adequate amounts are normally present in most groundwater.
Microbial growth and metabolism need an energy supply. Microorganisms obtain their energy for metabolism and biosynthesis either by converting sunlight into chemical energy (phototrophs) or extracting energy from organic or inorganic chemicals (chemotrophs). Oxidation-reduction reactions are the basis of all energy-producing reactions, and adenosine triphosphate (ATP) is the principle energy-transport molecule of the cell. Microorganisms then use this energy to perform specific functions, one of which is biosynthesis of new cell components. Energy can be extracted from substrates in one of three ways: respiration, fermentation, and anaerobic respiration. Aerobic bacteria use oxygen as a terminal electron acceptor and oxidize carbon substrate to CO
2
(i.e., respiration). Some anaerobic bacteria use inorganic molecules such as NO
3
−
, NO
2
−
, or SO
4
2−
as electron acceptors with CO
2
as the final carbon oxidation product (i.e., anaerobic respiration), while others use organic molecules such as pyruvate as an electron acceptor with fermentation products such as lactic acid as a final carbon oxidation product (i.e., fermentation). Facultative bacteria have the capability of growing in the presence or absence of oxygen.
Denitrifying bacteria, which are able to use nitrogen oxides as electron acceptors in place of oxygen, are essentially facultative bacteria. Obligate anaerobes, such as sulfate reducing bacteria often survive in the presence of facultative bacteria. There are two nutritional types of microorganisms—those that obtain their carbon for biosynthetic processes from organic compounds (i.e., heterotrophs) and those that obtain their carbon for biosynthetic processes from CO
2
(i.e., autotrophs). Most denitrifying bacteria and sulfate reducing bacteria are heterotrophic and few can grow autotrophically.
Nitrate pollution in groundwater is a common problem in all European and North American countries. Nitrate contamination often exceeds the maximum contaminant limit of 10 mg N/L, and poses a major threat to drinking water supplies. The standard was imposed because nitrate is linked to infant methemoglobinemia (“blue baby” syndrome). The formation of nitroso-compounds which are known carcinogens also has been linked to nitrate. Conventional nitrate treatment technologies include reverse osmosis and ion exchange. Activated carbon adsorption in conjunction with pH adjustment has also been used in experimental studies to successfully remove nitrate. Biological denitrification is a technology mainly studied for surface water treatment. Compared to conventional technologies, biological denitrification is very cost-effective and is promising for in situ remediation, particularly as practiced with the present invention.
The main biological processes involving inorganic nitrogen are shown in FIG.
1
. Nitrogen fixation involves the synthesis of cellular nitrogen compounds from elementary nitrogen. It is associated primarily with certain agricultural plants in which bacteria in a symbiotic or free living state. Deamination reactions are associated with the lysis of dying cells and the formation of ammonia from organic nitrogen compounds. Nitrification is the oxidation of NH
4
+
to nitrate, via nitrite, and is carried by nitrifying bacteria. With regard to nitrate metabolism, assimilation is defined as the conversion of nitrate to cellular organic nitrogen via ammonia, and dissimilation (or nitrate respiration) is defined as the oxidation of carbon compounds at the expense of nitrate which acts as the alternative electron acceptor to oxygen.
Denitrification is a special case of dissimilation in which gaseous nitrogens are end products. The principal products are nitrogen gas (N
2
) and nitrous oxide (N
2
O), though nitric oxide (NO) has occasionally been detected. During the denitrification process, nitrogen oxides serve as terminal electron acceptors instead of oxygen and are reduced by a unique suite of complex enzymes that conserve energy in several reductive steps by electron transport phosphorylation. The pathway of denitrification is thought to be:
NO
3
−
→NO
2
−
→NO(
g
)→N
2
O(
g
)→N
2
(
g
).
The reduction of nitrate to nitrite is known as denitratation, and the reduction of nitrite is called denitritation. Each reaction step involves a different enzyme. For example, nitrate reductase (NaR) catalyzes the reduction of nitrate to nitrite and nitrite reductase (NiR) catalyzes the reduction of nitrite to gaseous products.
Nitrite and N
2
O are often observed to accumulate temporarily during denitrification. This accumulation can often been explained by relative differences in reaction rates for the different steps in the sequence. For example, when denitritation rate is higher than denitratation rate, nitrite is reduced as soon as it appears and so, it does not accumulate in the system. But if denitratation is faster than denitritation, nitrite build-up will be noticed. Several reasons have been suggested to explain this phenomenon: evolution of the microbial population, enzymatic adaptation to changes in the environment (particularly, dissolved oxygen concentration and pH), inhibition of nitrite reductase, or effect of external carbon loading.
Denitrifying bacteria, which are able to use nitrogen oxides as electron acceptors in place of oxygen with the evolution of gaseous products, are biochemically and taxonomically very diverse. Most bacteria are heterotrophs and some utilize one-carbon compounds, whereas others grow autotrophically on H
2
and CO
2
or reduced sulfur compounds. One group is photosynthetic. Most have all of the reductases necessary to reduce NO
3
−
to N
2
, some lack NO
3
−
reductase and are termed NO
2
−
dependent, and others lack N
2
O reductase and thus yield N
2
O as the terminal product. Still other organisms possess N
2
O reductase but cannot produce N
2
O from NO
3
−
or NO
2
−
. Among the denitrifying bacteria, the genus Pseudomonas, which includes the most commonly isolated denitrifying bacteria from both soils and aquatic sediments, may represent the most active denitrifying bacteria in natural environments. Some of them are NO
2
−
dependent, and some strains produce N
2
O. Denitrifying pseudomonads include
P. denitrificans, P. fluorescens, P. stutzeri, P. aerogenes, P. aureofaciens, P. caryophylli
, and
P. chlororaphis.
Increasing the population of denitrifying bacteria is the ultimate goal of denitrification. Natural in situ biological denitrification, which is too slow to do efficient groundwater remediation, can be promoted by adding suitab
Lu Yongming
Nuttall Herbert E.
Myers Jeffrey D.
Redding David A.
University of New Mexico
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