Jürgen Warrelmann

Nucleotide sequence of the rpoN (hno) gene region of Alcaligenes eutrophus: evidence for a conserved gene cluster

The nucleotide sequence of the rpoN gene, formerly designated hno, and flanking DNA regions of the aerobic hydrogen bacterium Alcaligenes eutrophus has been determined; rpoN codes for the RNA polymerase sigma factor σ54 involved in nitrogen regulation and diverse physiological functions of gram-negative bacteria. In A. eutrophus hydrogen metabolism is under control of rpoN. The Tn5-Mob insertion in a previously isolated pleiotropic mutant was mapped within the rpoN gene. The derived amino acid sequence of the A. eutrophus RpoN protein shows extensive homology to the RpoN proteins of other organisms. Sequencing revealed four other open reading frames: one upstream (ORF280) and three downstream (ORF130, ORF99 and ORF > 54) of the rpoN gene. A similar arrangement of homologous ORFs is found in the rpoN regions of other bacteria and is indicative of a conserved gene cluster.

Genetics of hydrogenase from aerobic lithoautotrophic bacteria

Aerobic facultatively autotrophic hydrogen bacteria are distinguished on the basis of their hydrogen-oxidizing enzyme system (Hox). The major group, represented by Paracoccus denitrificans and Pseudomonas facilis, contains a membrane-bound, electron transport-coupled protein. Species of Nocardia are characterized by the possession of a cytoplasmic NAD-dependent hydrogenase. Both enzymes are present in strains of Alcaligenes. All hydrogenases from lithoautotrophs are H2-consuming nickel-iron-sulfur proteins. Despite these common characteristics, hydrogenases differ in catalytic and molecular properties, in particular in the regulation of enzyme synthesis. Hydrogenase formation is either inducible by H2 (e.g. P. denitrificans strain F1, Alcaligenes hydrogenophilus) or subject to derepression in response to the supply of reductant, temperature, and oxygen (e.g. Alcaligenes eutrophus). The only plasmid-encoded Hox function has been conclusively identified in species of Alcaligenes. Structural and regulatory hox genes reside on megaplasmids, ranging in size between 400 and 500 kilobase pairs (kb). Most of the plasmids are self-transmissible by conjugation. Hox genes of A. eutrophus H16 have been localized by plasmid curing, genetic transfer, molecular cloning and analysis of plasmid deletions and insertions. They seem to be clustered in a DNA sequence of approximately 50 kb, representing several transcriptional units. In addition, a chromosomally encoded regulatory function is required for the expression of plasmid-linked hox genes. Plasmid pHGl of A. eutrophus H16 has been transferred to the non-lithoautotrophic soil bacterium JMP222. Both hydrogenases are expressed in the new host. The current state of hydrogenase genetics in Alcaligenes is discussed in reference to hydrogenase systems of other lithoautotrophic bacteria.

In-Situ Phytoremediation of TNT-Contaminated Soil

Parts of the area of the derelict World-War-II ordnance plant Werk Tanne (Clausthal-Zellerfeld, Harz, Germany) are heavily contaminated by chemicals resulting from TNT production and particularly by TNT itself. High soil contamination has to be treated with ex-situ methods but for the extended contamination of surface soil, in-situ phytoremediation is appropriate. The TNT-degrading potential of the rhizosphere of the planted trees and shrubs themselves is augmented by highly active mycorrhiza and white-rot fungi. A phytoremediation measure was established to scale with heavy machinery (soil grader), including the incorporation of white-rot fungi into the soil and planting of mycorrhized trees and shrubs. The effects of site preparation, mycorrhized rhizosphere and white-rot fungi on the degradation of TNT were assessed over one year using a complex monitoring scheme, including a battery of five biotests and field investigations of selected indicators (soil mesofauna, decomposition). The results of the monitoring showed the great influence of the grading procedure for site preparation, a diversified sensitivity of the biotest battery and complex reactions of the field indicators. The grading procedure effectively reduced the contamination (almost 90% within the first six months regardless of the initial levels). The phytoremediation measure as a whole reduced hazards of transport of nitro-aromatics by dust or leachate, initiated a secondary succession of the soil ecosystem that could transform the remaining TNT and metabolites over a longer period of time, and thus proved to be an effective decontamination measure applicable in large-scale technology.

Biological Treatment of TNT-Contaminated Soil by a Two-Stage Anaerobic/Aerobic Process

TNT is found as a major soil contaminant at the sites of armament plants from World War I and II. While no complete biological mineralization of TNT has yet been found, many bacteria can co-metabolically reduce the nitro groups of TNT to amino groups during anaerobic fermentation of readily available substrates. In a soil slurry, the final reduction product (TAT) irreversibly reacts with soil components. The biological treatment of TNT-contaminated soil with complete reduction of TNT to TAT and immobilization of TAT is a potential remediation process that is currently being tested at a technical scale. The large-scale experiment is accompanied by investigations on the immobilization of TAT and the toxicity of the treated soil.

Mutants of pseudomonas facilis defective in lithoautotrophy

SUMMARY: Mutants of Pseudomonas facilis impaired in lithoautotrophic metabolism (Aut-) were obtained by Tn5 mutagenesis, mitomycin C treatment or incubation at sublethal temperature. A colony assay provided a fast screening method for hydrogenase-negative mutants. Three classes of mutants were identified: the first had lost the ability to grow autotrophically with H2 and heterotrophically with NO3 - as nitrogen source; the second lacked both hydrogenase and ribulose-1,5-bisphosphate carboxylase activity but nitrate metabolism was unaffected; the third retained the activities of the key enzymes of lithoautotrophic metabolism but failed to grow with H2 and CO2. All of the mutants showed the plasmid pattern of the wild-type.

Genetic transfer of lithoautotrophy mediated by a plasmid-cointegrate from Pseudomonas facilis

Pseudomonas facilis (DSM 620) is host of two plasmids one of which (pHG22-a) has been shown to be involved in lithoautotrophic metabolism. The lithoautotrophic marker was transferred via conjugation to mutants of two wild type strains of P. facilis and to the heterotrophic bacterium Pseudomonas delafieldii. The transfer required mobilization by the IncP1 plasmid RP4. Transconjugants contained a plasmid which neither correlated in size with RP4 nor with pHG22-a. This newly formed plasmid, pHG22-c, was shown to be a cointegrate consisting of RP4 DNA and a 50-kb insert derived from the native plasmid pHG22-a. DNA-DNA hybridization using lithoautotrophic genes of Alcaligenes eutrophus as DNA probes, revealed the presence of hydrogenase structural and regulatory genes in addition to genes of autotrophic carbon dioxide fixation on the cointegrate pHG22-c.

Erprobung und Erfolgskontrolle eines Phytoremediations-verfahrens zur Sanierung Sprengstoff-kontaminierter Böden

ZusammenfassungDie Befunde des in seiner Konzeption in Teil I dargestellten Freilandexperimentes dokumentieren die TNT-Abreicherung und Erholung des Standortes im Laufe der Phytoremediation. Das Monitoring umfasst Erhebungen im Feld (Nitroaromatengehalte in Boden, Sickerwasser und Pflanzen; Erfassung von Bodenorganismen und Streuabbau) und den Einsatz einer Biotestbatterie mit 6 Einzeltests. Die Daten belegen die hohe Heterogenität der TNT-Kontamination, eine rasche initiale Abreicherung und eine geringe Verlagerung von Nitroaromaten in Pflanzen und Sickerwasser. Die anfänglich rasche Transformation des TNT zu Aminodinitrotoluolen lässt nach 3 Monaten deutlich nach. Mit Hilfe der Biotestbatterie wird die Toxizität des Bodens differenziert beurteilbar; die boden-biologischen Befunde lassen Langzeitwirkungen der TNT-Belastung wahrscheinlich werden. Eine Gesamtbeurteilung des Projekts wird im dritten Teil der Artikelserie erfolgen.AbstractResults of a field experiment (please refer to Warrelmann et al. 2000a) document TNT-degradation and regeneration after the establishment of a phytoremediation. Various approaches are included in a complex monitoring system, which are conducted in the field (nitroaromatics in soil, seepage water, and plants; soil fauna; soil fungi; decomposition) as well as in the lab (a battery of 6 biotests). The data document a high heterogeneity of the TNT-contamination, a rapid initial decrease and a mediocre displacement of nitroaromatics in plants and seepage water. The initial rapid transformation of TNT to aminodinitrotoluenes ceases significantly after 3 months. Results of the biotest battery allow a sophisticated assessment of soil toxicity; the results from the soil ecological field investigations make the long-term effects of TNT-contamination likely. In a third part of the series of articles, a synopsis and judgement of the project will be delivered.

Pilot Plant Stage Bioremediation of CKW- and BTEX-Contaminated Soil by in Situ Infiltration in Combination with on-Site Water and Air Treatment at the Model Site Eppelheim

The site at Eppelheim is an abandoned dump area with chloroethenes and BTEX aromatics as the main contaminants. It has been selected for the model site development program of the state of Baden-Wurttemberg (Germany) to establish bioremediation processes for the on-site or in-situ treatment of soil, water and air, carrying mixed contaminations, and to develop them into the pilot plant stage. The bioremediation of CKW- and BTEX-contaminated soil by in-situ infiltration in combination with on-site water treatment in solid bed bioreactors and air treatment in biofilters turned out to be the most effective process. Application in the field revealed a reduction of the initial load of contaminants in the soil by 94.5 %. In the bioreactors, 99 % of the contaminants were eliminated, and at least 80% of them were mineralized to CO2 and CH4.

The Bioremediation Model Site at Eppelheim

The development of new, integrated and fully documented techniques for the bioremediation of soil, water and air contaminated with volatile chlorinated hydrocarbons at the model site near the city of Eppelheim was conducted within the framework of the model site program established by the Ministry of the Environment of the State of Baden Wurttemberg through the State Authority for Environmental protection and funded by the “Kommunale Altlastenfond”.