Parathion also contains a thioester linkage that is analogous to the chemical structure of several chemical warfare agents, including sarin. Consequently, parathion is an excellent model for studying enhanced biodegrada–tion of environmental contaminants. Mineralization of parathion requires three unique catabolic properties: hydrolysis of parathion, mineralization of p-nitrophenol, and mineralization of diethyl thiophos–phate . The objective of this research was to develop a coculture capable of hydrolyzing parathion and degrading its metabolite p-nitrophenol; evaluate the kinetics of the reaction; and test the suitability of the coculture for use in a flow-through biofilm reactor for parathion bio–degradation.Escherichia coli strain SD2 was constructed by introducing plasmid pWM513, harboring the genes for parathion hydrolysis and ampicillin resistance, and plasmid pMAG1, carrying the green fluorescent pro–tein gene and tetracycline resistance, into Escherichia coli DH10B. The plasmids were inserted by electropo–ration and strain SD2 was selected using media containing both ampicillin and tetracycline. Strain SD2 was used together with Pseudomonas putida KT2440 carrying plasmid pPNP, harboring the genes for p–nitrophenol degradation and also tetracycline resistance. Strain KT2440 is naturally resistant to ampicillin, so the two strains could be cultured in media containing both antibiotics, and consequently maintained the genes required to degrade parathion. The ability of the coculture to degrade 0.5 mM parathion was evaluated during growth of the strains in a minimal medium containing glucose as the carbon source. P-nitrophenol accumulated in the medium during the growth of strain SD2 alone,drainage gutter resulting from parathion hydrolysis .
In contrast, the accumulation of p-nitrophenol was only transient in the coculture, as a result of the bio–degradation activity of strain KT2440. Kinetic analysis indicated that 2 mM p–nitrophenol was fully inhibitory to the growth of the coculture; consequently, this concentration represented the upper limit for parathion bio–degradation . The coculture was used to cultivate a biofilm in a par–allel plate flow cell for imaging by confocal microscopy. After 72 hours of growth in continuous mode, the biofilm was stained with a red fluores–cent nucleic acid dye and imaged using a confocal microscope. The dye caused the Pseudomonas strain to appear red, while the Escherichia coli strain appeared green and yellow as a result of the colocalization of green color fluorescing from its green fluorescent protein. The images indicated that the biofilm was dominated by Pseudomonas, although E. coli was stably maintained. The results suggest that the two strains could be used as part of a flow-through biofilm reactor for detoxification of parathion. Several industries in the United States and Puerto Rico are dedicated to the manufacture of filters of different materials, including nitrocellulose . These filters are very important in ensuring the purity of ingredients in food and pharmaceutical industries. However, the nitro–cellulose used for manufacturing these filters is considered a hazardous waste for its ignitability , high flammability and oxidizing properties. The Department of Defense also has sites contaminated with NC since it is a major component in explosives. Nitrocellulose exhibits good chemical stability, which for years made incineration the preferred method for dis–posing of the NC wastes. Alkaline hydrolysis appears to be a rapid process for nitrocellulose degradation, but research is limited. This investigation involves the eluci–dation of the mechanism of alkaline hydrolysis and its combination with biodegradation.Table 1 shows the research methods and tools employed in this study. Every step in Table 1 seems to play an important role for either hydrolysis or bio–degradation itself. Interpretation of the species left after degradation time facilitates analysis. Comparison between liquid and agar media is important to really determine if physical attachment is another factor for better fungus growth. Figure 1 shows a possible precipitation of products after acidification on the 9 mL NaOH treatment.
Figure 2 shows fungus growth from 10 mcL of spores suspen–sion placed on a petri dish containing NC as sole carbon source. HPLC assays have been made on a growth curve in a culture tube . A total of 0.1 g of NC hydrolyzed in 6 mL of SSC buffer was used. The strongest peaks are shown. The sample represents one-third of the total area. A total of 20 mcL were injected in a 50:50 acetonitrile:water mixture. Assays are being done using wavelengths of 210 nm and 214 nm, due to the abundance of probably dicarboxilic acids in the hydrolyzed NC.Polycyclic aromatic hydrocarbons are a con–cern in the environment because they are toxic and carcinogenic. Polycyclic aromatics are more recalci–trant in the soil than other hydrocarbons because they are hydrophobic and tend to migrate into the soil . Most PAHs occur as a result of fossil fuel combustion; thus, high concentra–tions of PAHs are found at the sites of active and inactive oil refineries . This study focuses on oil and petroleum contami–nated soil samples taken from Alameda Naval Air Station at Alameda Point, Calif. Pacific Coast Oil Works refinery used the site between 1879 and 1903. After the refinery closed in the 1930s, the U.S. Army and then the U.S. Navy owned the property. In 1991, jet fuel spilled from the jet engine test facility on the site. Heavy rains resulted in jet fuel in the overflow of storm drains. Damage to the storm drains during the Loma Prieta earthquake in 1989 may have caused ground water contamination. Recent studies showed total petroleum hydrocarbon amounts rang–ing from 100 to 10,000 mg/kg soil. Due to the long term and high concentration of oil in the soil it is likely that there are microorganisms capable of degrading the oil products. This study will examine the use of Inipol EAP 22, the same fertilizer used in the Exxon Valdez oil spill clean-up, to obtain optimal growth conditions of the naturally occurring bacteria using CO2 output to monitor the degradation of PAHs and hydrocarbons. Inipol EAP 22 is particularly attractive for this site since it is oleophilic and should make the strongly sorbed PAH components more bio–available, thereby stimulating bio–degradation.More than 8,000 Department of Defense sites need clean-up efforts .
TNT was the most commonly occurring compound within the contaminated samples from these sites. Fortunately, TNT is biodegradable, making in situ bio–remediation a cost–effective and rapid alternative for site cleanup. The toxicity of nitroarenes and their metabolites have been studied in a variety of biological systems but we have been unable to find any studies related to chemotaxis of nitroaromatic compounds in the scientific literature. Chemotaxis allows bacteria to respond to chemical gradients, seeking higher levels of potential nutrients and lower levels of inhibitors. Organisms that have developed mechanisms with which to beneficially orient themselves with respect to these gradients of different types may have a competitive advantage over other organisms. Also, the value of motility as a survival factor for bacteria in environments where nutrients or harmful agents are discontinuously dis–tributed, e.g., contaminated sites, seems obvious. The present study will target how bacteria can use nitroaromatic compounds as a source of nutrients and as a dispersal mechanism in soil,large square pots especially as it may apply to in situ bio–remediation.Chemotaxis provides a means for bacteria to respond to environmental gradients of potential nutrients and toxins, resulting in direct motility towards or away from these substances . The P. fluorecens bacterial strain used responded impressively according to our predictions . This is the first time that bacteria have been demonstrated to be attracted to explosive compounds. The strong attraction of soil bacteria to DNT was also verified using soil perfusion column leachate from field samples taken at Ft. Ord, Calif. In these leachate tests, the chemotactic indices observed for DNT are some of the highest rates ever observed for any type of chemotaxis, including the ones observed for P. fluorecens for concentrations of 2,4 and 2,6-dinitrotoluene that ranged from 0.12 M to 1.20×10-3 M. The results of this work have important implications for the ecology of TNT-degrading bacteria; it also may suggest ways that micro–environments containing explosives might be controlled to increase bio–degradation rates in situ.More than 8,000 Department of Defense sites need clean-up efforts . In a compilation of soil samples collected from 44 Army ammunition plants, arsenals and depots, 28% contained detectable levels of explosives. TNT was the most commonly occurring compound within the contaminated samples, and was seen in 66% of those samples. Facilities that may be contaminated with explosives include manufacturing plants, ordnance works, Army ammunition plants, ammunition depots, Army and Naval proving grounds, burning grounds, artillery impact ranges, explosives disposal sites, bombing ranges, firing ranges, and ordnance test and evaluation facilities.
Due to its toxicity and recalcitrance, the U.S. Environmental Protection Agency has listed TNT as a priority pollutant . Bio-remediation of organic contaminated soils has proven to be one of the fastest and cheapest remediation technologies available. TNT and its daughter products are highly recalcitrant, especially in highly weathered soils, i.e., soils that have been exposed to the environment for a number of years under sub–optimal microbial activity conditions. The present study examines weathered and unweathered soils and looks at the ability of a number of bio–stimulants to increase total microbial respiration.Two-gallon buckets were converted to soil bioreactors . These reactors use the same CO2 trapping principle as the bio–meter flasks, but allow for a much greater soil sample. The KOH trap was a funnel plugged at the bottom with a rubber stopper, which was held in place with epoxy. These traps rested on top of the soil, and could hold up to 80 ml of solution. Larger ascarite-top filters were made using 50 ml Corning vials. A hole was drilled into the bottom of each vial, through which a piece of plastic tubing would fit. This tube was connected to a stopcock, which let the filtered air into the bucket during the times when the KOH was changed. The attached diagram shows the bio–meter bucket design. The soil in each bucket was mixed well prior to the respiration measurements. After the bucket was sealed, the KOH was added to its trap, and the buckets sat for four days. In this four-day period, the KOH was changed and titrated as necessary, with intervals from an hour to a day, depending on the rate of respiration in the soil samples. These intervals change because a faster respiration rate can create enough CO2 to saturate the KOH solution. Therefore, the KOH monitoring a more active soil sample must be titrated much more often than in a slowly respiring sample. For each bucket, three experiments were run: an unamended control, where the soil was taken directly from the buckets in which it was collected, and lasting 3.57 days, an inorganic nutrient amendment, where 1000 ml of MSM was added to each bucket, and lasting 2.93 days, and an organic nutrient amendment, where an aqueous molasses solution was added to each bucket, and lasting 4.80 days. Respiration was measured daily and samples for HPLC analyses of contaminants were taken initially and at the end of the treatment.The respiration analysis indicates that unweathered soil responds faster and in greater magnitudes to nutrient amendments . The results suggest that the weathered soils may have stressed populations and cannot react as quickly to the amendments, especially because they actually reduce their respiration rate in response to MSM addition. The molasses-amended samples all respired at a greater rate than with the other amendments. This result implies that molasses may be used as an effective nutrient source to increase bacterial activity. Three of the four molasses-amended samples are among the lowest four in concentrations of the suspected contaminant located by the HPLC. The HPLC results also suggest a correlation between bacterial activity and bio–degradation. Glycerol trinitrate , also known as nitroglyc–erin, is extensively utilized for the production of explosives and pharmaceuticals. GTN is a hazardous waste product of increasing environmental concern. Current disposal techniques, such as open-air burning and incineration, are expensive and can produce hazardous waste byproducts. Bio–remediation systems could remediate explosive contaminants at approximately a tenfold lower treatment cost and with increased public acceptance. An ecological investigation of GTN-contaminated sites at the Naval Surface Warfare Station in Maryland resulted in the isolation of a Bacillus thuringiensis/cereus strain able to degrade GTN. HPLC and TLC analysis by other researchers of GTN metabolism in cell-free systems suggested that there was a sequential denitration to dinitrate isomers, mononitrate isomers and ultimately to glycerol.