Microbial transformation of 2,4,6-trinitrotoluene in aerobic soil columns.

2,4,6-Trinitrotoluene (TNT)-contaminated soil material of a former TNT production plant was percolated aerobically in soil columns. Nineteen days of percolation with a potassium phosphate buffer supplemented with glucose or glucose plus ammonium sulfate caused an over 90% decline in the amount of extractable nitroaromatics in soils containing 70 to 2,100 mg of TNT per kg (dry weight). In the percolation solution, a complete elimination of TNT was achieved. Mutagenicity and soil toxicity were significantly reduced by the percolation process. 4-N-Acetylamino-2-amino-6-nitrotoluene was generated in soil and percolation fluid as a labile TNT metabolite.

Bioremediation of 2,4,6-trinitrotoluene-contaminated soils by two different aerated compost systems.

Two composting systems were compared on a laboratory scale as a bioremediation technology for degradation or immobilization of 2,4,6-trinitrotoluene (TNT) in contaminated soils. The first compost was aerated from the beginning whereas the second compost was only aerated after an anaerobic prephase of 65 days. In the first compost system the TNT concentration declined rapidly by 92% but, at the end, TNT could be partially recovered. During the anaerobic prephase of the second compost system, TNT was almost completely converted to aminodinitrotoluenes, which during the subsequent aeration almost entirely disappeared. In addition, the second compost generated less toxic material than the first one as confirmed by inhibition of bioluminescence of Vibrio fischeri. These data show that microbiological TNT-degradation systems can be successfully designed which are prerequisite for an efficient bioremediation of contaminated soils.

Microbial degradation of explosives and related compounds.

The pollution of soil and water with explosives and related compounds caused by military activities has been known for a long time, but progress in understanding the environmental fate of such substances has only been made in the last few years. Microbial processes could be used for the remediation of explosives-contaminated soils and waste waters because it has been shown that a variety of different microorganisms are able to metabolize these chemical compounds. In some cases even a complete mineralization has been found, whereas in others only biotransformation reactions took place, producing more or less toxic and/or recalcitrant metabolites. Studies with pure cultures of bacteria and fungi have given detailed insights into the biodegradation pathways of at least some nitroorganic compounds. Additionally, some of the key enzymes have been isolated and purified or studied in crude extracts. This review summarizes information on the biodegradation and biotransformation pathways of several important explosives. This may be useful in developing microbiological methods for a safe and economic clean-up of soil and water contaminated with such compounds. It also shows the necessity of further investigations concerning the microbial metabolism of these substances.

N5,N10-methenyltetrahydromethanopterin cyclohydrolase from the extremely thermophilic sulfate reducing Archaeoglobus fulgidus: comparison of its properties with those of the cyclohydrolase from the extremely thermophilic Methanopyrus kandleri.

Archaeoglobus fulgidus and Methanopyrus kandleri are both extremely thermophilic Archaea with a growth temperature optimum at 83 degrees C and 98 degrees C, respectively. Both Archaea contain an active N5,N10-methenyltetrahydromethanopterin cyclohydrolase. The enzyme from M. kandleri has recently been characterized. We describe here the purification and properties of the enzyme from A. fulgidus. The cyclohydrolase from A. fulgidus was purified 180-fold to apparent homogeneity and its properties were compared with those recently published for the cyclohydrolase from M. kandleri. The two cytoplasmic enzymes were found to have very similar molecular and catalytic properties. They differed, however, significantly with respect of the effect of K2HPO4 and of other salts on the activity and the stability. The cyclohydrolase from A. fulgidus required relatively high concentrations of K2HPO4 (1 M) for optimal thermostability at 90 degrees C but did not require salts for activity. Vice versa, the enzyme from M. kandleri was dependent on high K2HPO4 concentrations (1.5 M) for optimal activity but not for thermostability. Thus the activity and structural stability of the two thermophilic enzymes depend in a completely different way on the concentration of inorganic salts. The molecular basis for these differences are discussed.

Formylmethanofuran: tetrahydromethanopterin formyltransferase and N5,N10-methylenetetrahydromethanopterin dehydrogenase from the sulfate-reducing Archaeoglobus fulgidus: similarities with the enzymes from methanogenic Archaea.

The sulfate-reducing Archaeoglobus fulgidus contains a number of enzymes previously thought to be unique for methanogenic Archaea. The purification and properties of two of these enzymes, of formylmethanofuran: tetrahydromethanopterin formyltransferase and of N5,N10-methylenetetrahydromethanopterin dehydrogenase (coenzyme F420 dependent) are described here. A comparison of the N-terminal amino acid sequences and of other molecular properties with those of the respective enzymes from three methanogenic Archaea revealed a high degree of similarity.

Salt dependence, kinetic properties and catalytic mechanism of N-formylmethanofuran:tetrahydromethanopterin formyltransferase from the extreme thermophile Methanopyrus kandleri.

N-Formylmethanofuran(CHO-MFR):tetrahydromethanopterin(H4MPT) formyltransferase (formyltransferase) from the extremely thermophilic Methanopyrus kandleri was purified over 100-fold to apparent homogeneity with a 54% yield. The monomeric enzyme had an apparent molecular mass of 35 kDa. The N-terminal amino acid sequence of the polypeptide was determined. The formyltransferase was found to be absolutely dependent on the presence of phosphate or sulfate salts for activity. The ability of salts to activate the enzyme decreased in the order K2HPO4 > (NH4)2SO4 > K2SO4 > Na2SO4 > Na2HPO4. The salts KCl, NaCl and NH4Cl did not activate the enzyme. The dependence of activity on salt concentration showed a sigmoidal curve. For half-maximal activity, 1 M K2HPO4 and 1.2 M (NH4)2SO4 were required. A detailed kinetic analysis revealed that phosphates and sulfates both affected the Vmax rather than the Km for CHO-MFR and H4MPT. At the optimal salt concentration and at 65 degrees C, the Vmax was 2700 U/mg (1 U = 1 mumol/min), the Km for CHO-MFR was 50 microM and the Km for H4MPT was 100 microM. At 90 degrees C, the temperature optimum of the enzyme, the Vmax was about 2.5-fold higher than at 65 degrees C. Thermostability as well as activity of formyltransferase was dramatically increased in the presence of salts, 1.5 M being required for optimal stabilization. The efficiency of salts in protecting formyltransferase from heat inactivation at 90 degrees C decreased in the order K2HPO4 = (NH4)2SO4 >> KCl = NH4Cl = NaCl >> Na2SO4 > Na2HPO4. The catalytic mechanism of formyltransferase was determined to be of the ternary-complex type. The properties of the enzyme from M. kandleri are compared with those of formyltransferase from Methanobacterium thermoautotrophicum, Methanosarcina barkeri and Archaeoglobus fulgidus.

Methyl-coenzyme M reductase and other enzymes involved in methanogenesis from CO2 and H2 in the extreme thermophile Methanopyrus kandleri.

Methanopyrus kandleri belongs to a novel group of abyssal methanogenic archaebacteria that can grow at 110 degrees C on H2 and CO2 and that shows no close phylogenetic relationship to any methanogen known so far. Methyl-coenzyme M reductase, the enzyme catalyzing the methane forming step in the energy metabolism of methanogens, was purified from this hyperthermophile. The yellow protein with an absorption maximum at 425 nm was found to be similar to the methyl-coenzyme M reductase from other methanogenic bacteria in that it was composed each of two alpha-, beta- and gamma-subunits and that it contained the nickel porphinoid coenzyme F430 as prosthetic group. The purified reductase was inactive. The N-terminal amino acid sequence of the gamma-subunit was determined. A comparison with the N-terminal sequences of the gamma-subunit of methyl-coenzyme M reductases from other methanogenic bacteria revealed a high degree of similarity. Besides methyl-coenzyme M reductase cell extracts of M. kandleri were shown to contain the following enzyme activities involved in methanogenesis from CO2 (apparent Vmax at 65 degrees C): formylmethanofuran dehydrogenase, 0.3 U/mg protein; formyl-methanofuran:tetrahydro-methanopterin formyltransferase, 13 U/mg; N5,N10-methylenetetrahydromethanopterin cyclohydrolase, 14U/mg; N5,N10-methenyltetrahydromethanopterin dehydrogenase (H2-forming), 33 U/mg; N5,N10-methylenetetrahydromethanopterin reductase (coenzyme F420 dependent), 4 U/mg; heterodisulfide reductase, 2 U/mg; coenzyme F420-reducing hydrogenase, 0.01 U/mg; and methylviologen-reducing hydrogenase, 2.5 U/mg. Apparent Km values for these enzymes and the effect of salts on their activities were determined. The coenzyme F420 present in M. kandleri was identified as coenzyme F420-2 with 2-gamma-glutamyl residues.

Formylmethanofuran: tetrahydromethanopterin formyltransferase from Methanosarcina barkeri. Identification of N5-formyltetrahydromethanopterin as the product.

Formylmethanofuran: tetrahydromethanopterin formyltransferase was purified from methanol grown Methanosarcina barkeri to apparent homogeneity and characterized with respect to its molecular and kinetic properties. The enzyme was found to be very similar to the formyltransferase from H2/CO2 grown Methanobacterium thermoautotrophicum. It also catalyzed the formation of N5-formyltetrahydromethanopterin rather than of N10-formyltetrahydromethanopterin from formylmethanofuran and tetrahydromethanopterin.

N-furfurylformamide as a pseudo-substrate for formylmethanofuran converting enzymes from methanogenic bacteria.

Methanofuran (4-[N-(4,5,7-tricarboxyheptanoyl-gamma-L-glutamyl)-gamma-L- glutamyl)-p-(beta-aminoethyl)phenoxymethyl]-2-(aminomethyl)furan is a coenzyme involved in methanogenesis. The N-formyl derivative is an intermediate in the reduction of CO2 to CH4 and the disproportionation of methanol to CO2 and CH4. Formylmethanofuran dehydrogenase and formylmethanofuran:tetrahydromethanopterin formyltransferase are the enzymes catalyzing its conversions. We report here that the two enzymes from Methanosarcina barkeri and the formyltransferase from Methanobacterium thermoautotrophicum can also use N-furfurylformamide as a pseudo-substrate albeit with higher apparent Km and lower apparent Vmax values. N-Methylformamide, formamide, and formate were not converted indicating that the furfurylamine moiety of methanofuran is the minimum structure required for the correct binding of the coenzyme.