Albin Alfreider

Groundwater samples do not reflect bacterial densities and activity in subsurface systems

Total cell numbers, abundance of respiring bacteria and [3H]thymidine and [14C]leucine incorporation rates were investigated in four groundwater wells of low nutrient content. Total cell numbers in the pumped groundwater were low (14 × 103 to 279 × 103 cells mL−1), and [3Hthymidine and [14C]leucine incorporation rates were, with one exception, below the detection limit. Therefore we exposed sediments in situ for 2 months which allowed us to determine bacterial numbers and incorporation rates of labeled substrates by bacteria attached to sediment particles. The two habitats differed considerably in all bacterial parameters both in magnitude and seasonal trends. Total bacterial numbers of sandy sediments (52.1 ± 21.3 × 106 cells cm−3) corresponded in average to 663 cm3 of pumped groundwater (78.5 ± 61.5 × 106 cells L−1). For the fraction of respiring bacteria this ratio was on average 3032 cm3 (sediments: 10.3 ± 5.4 × 106 respiring cells cm−3; groundwater: 3.39 ± 6.01 × 106 respiring cells L−1). The percentage of respiring bacteria in sandy sediments was between 6.0 and 41.4% (average 19.8) compared to 1.0 to 24.9% (average 5.23) in the pumped groundwater. Our results stress the importance of studying the microbial communities attached to sediment, as pumped groundwater samples may not be representative for the real structure and dynamics of microbial assemblages in subsurface environments.

Microbial Diversity in an in situ Reactor System Treating onochlorobenzene Contaminated Groundwater as Revealed by 16S Ribosomal DNA Analysis

A molecular approach based on the construction of 16S ribosomal DNA clone libraries was used to investigate the microbial diversity of an underground in situ reactor system filled with the original aquifer sediments. After chemical steady state was reached in the monochlorobenzene concentration between the original inflowing groundwater and the reactor outflow, samples from different reactor locations and from inflowing and outflowing groundwater were taken for DNA extraction. Small-subunit rRNA genes were PCR-amplified with primers specific for Bacteria, subsequently cloned and screened for variation by restriction fragment length polymorphism (RFLP). A total of 87 bacterial 16S rDNA genes were sequenced and subjected to phylogenetic analysis. The original groundwater was found to be dominated by a bacterial consortium affiliated with various members of the class of Proteobacteria, by phylotypes not affiliated with currently recognized bacterial phyla, and also by sporulating and non-sporulating sulfate-reducing bacteria. The most occurring clone types obtained from the sediment samples of the reactor were related to the beta-Proteobacteria, dominated by sequences almost identical to the widespread bacterium Alcaligenes faecalis, to low G+C gram-positive bacteria and to Acidithiobacillus ferrooxidans (formerly Thiobacillus ferrooxidans) within the gamma subclass of Proteobacteria in the upper reactor sector. Although bacterial phylotypes originating from the groundwater outflow of the reactors also grouped within different subdivisions of Proteobacteria and low G+C gram-positive bacteria, most of the 16S rDNA sequences were not associated with the sequence types observed in the reactor samples. Our results suggest that the different environments were inhabited by distinct microbial communities in respect to their taxonomic diversity, particular pronounced between sediment attached microbial communities from the reactor samples and free-living bacteria from the groundwater in- and outflow.

Diversity of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Large-Subunit Genes from Groundwater and Aquifer Microorganisms

To test our hypothesis that microbial autotrophic CO2 fixation plays an important role in subsurface systems of two large groundwater remediation projects, several anaerobic/microaerobic aquifer and groundwater samples were taken and used to investigate the distribution and phylogenetic diversity of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) large-subunit genes. Two primer sets were designed for amplifying partial-subunit genes of RubisCO forms I and II from the DNA, directly extracted from the samples. PCR products were used to construct five clone libraries with putative RubisCO form I sequences, and two libraries of DNA amplified by form II primers. Selected clones were screened for variation by restriction fragment length polymorphism analysis, and a total of 28 clone inserts were sequenced and further analyzed. The phylogenies constructed from amino acid sequences derived from the partial RubisCO large-subunit sequences showed a distinct pattern. Diverse sequences affiliated to the cluster of green-like type IA RubisCO sequences were found, representing various obligate and facultative chemolithoautotrophic Proteobacteria, whereas type II RubisCO sequences detected were most closely related to those of thiobacilli species. An isolate obtained from aquifer enrichment culture, which has been provisionally named Halothiobacillus sp. RA13 on the basis of its 16S rDNA sequence, was found to contain both types of RubisCO genes, i.e., forms I and II. Physiological and ecological considerations are discussed in the context of additional microbial data and physicochemical properties.

Bioremediation of chlorobenzene-contaminated ground water in an in situ reactor mediated by hydrogen peroxide.

New in situ reactive barrier technologies were tested nearby a local aquifer in Bitterfeld, Saxonia-Anhalt, Germany, which is polluted mainly by chlorobenzene (CB), in concentrations up to 450 microM. A reactor filled with original aquifer sediment was designed for the microbiological remediation of the ground water by indigenous bacterial communities. Two remediation variants were examined: (a) the degradation of CB under anoxic conditions in the presence of nitrate; (b) the degradation of CB under mixed electron acceptor conditions (oxygen+nitrate) using hydrogen peroxide as the oxygen-releasing compound. Under anoxic conditions, no definite degradation of CB was observed. Adding hydrogen peroxide (2.94 mM) and nitrate (2 mM) led to the disappearance of CB (ca. 150 microM) in the lower part of the reactor, accompanied by a strong increase of the number of cultivable aerobic CB degrading bacteria in reactor water and sediment samples, indicating that CB was degraded mainly by productive bacterial metabolism. Several aerobic CB degrading bacteria, mostly belonging to the genera Pseudomonas and Rhodococcus, were isolated from reactor water and sediments. In laboratory experiments with reactor water, oxygen was rapidly released by hydrogen peroxide, whereas biotic-induced decomposition reactions of hydrogen peroxide were almost four times faster than abiotic-induced decomposition reactions. A clear chemical degradation of CB mediated by hydrogen peroxide was not observed. CB was also completely degraded in the reactor after reducing the hydrogen peroxide concentration to 880 microM. The CB degradation completely collapsed after reducing the hydrogen peroxide concentration to 440 microM. In the following, the hydrogen peroxide concentrations were increased again (to 880 microM, 2.94 mM, and 880 microM, respectively), but the oxygen demand for CB degradation was higher than observed before, indicating a shift in the bacterial population. During the whole experiment, nitrate was uniformly reduced during the flow path in the reactor.

Expression of Chlorocatechol 1,2-Dioxygenase and Chlorocatechol 2,3-Dioxygenase Genes in Chlorobenzene-Contaminated Subsurface Samples

ABSTRACT In order to evaluate the in situ degradative capabilities of microorganisms in an underground reactor facility housing two flowthrough columns filled with aquifer soil, we examined the distribution and phylogeny of gene transcripts encoding enzymes capable of catalyzing the cleavage of the chlorinated aromatic ring during transformation of the main pollutant, chlorobenzene. Initial biostimulation of the autochthonous bacteria in the originally anaerobic reactor columns was achieved by injecting nitrate and oxygen in the form of H2O2. Two broad-range primer pairs were used for reverse transcriptase PCR (RT-PCR) of partial subunit genes of chlorocatechol 1,2-dioxygenase and catechol 2,3-dioxygenase from RNA directly extracted from different groundwater and aquifer samples. Samples retrieved from the lowermost sections of the reactor columns, which were operated in upflow mode, were positive for the presence of chlorocatechol 1,2-dioxygenase and catechol 2,3-dioxygenase mRNA. On the other hand, chlorocatechol 1,2-dioxygenase RT-PCR products were detected in a larger part of each reactor column, up to a zone 5.5 m above the bottom. Phylogenetic analyses of these chlorocatechol 1,2-dioxygenase sequences clearly separated them into two main clusters, one of which was closely affiliated with the broad-spectrum chlorocatechol 1,2-dioxygenase from Pseudomonas chlororaphis RW71. Analysis of sequences obtained from RT-PCR products amplified with catechol 2,3-dioxygenase primers revealed that their closest relative was the chlorocatechol 2,3-dioxygenase gene cbzE from Pseudomonas putida GJ31 (A. E. Mars, J. Kingma, S. R. Kaschabek, W. Reineke, and D. B. Janssen, J. Bacteriol. 181:1309-1318, 1999), with sequence similarities between 97.8 and 99.0%.

Distribution and diversity of autotrophic bacteria in groundwater systems based on the analysis of RubisCO genotypes.

A molecular approach, based on the detection of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) large subunit genes, was applied to investigate the distribution and diversity of autotrophic bacteria in groundwater systems. DNA extracts from 48 sampling stations, including a variety of pristine and polluted, shallow and deep-subsurface groundwater samples obtained from Germany and Austria, served as a template for the PCR amplification of form I (cbbL) and form II (cbbM) large subunit RubisCO genes. The majority of the samples (>80%) contained two different forms of RubisCO. In 17 samples, all three forms of RubisCO were identified. PCR products from four selected groundwater habitats containing all three forms of RubisCO were used to construct clone libraries. Based on restriction fragment length polymorphism (RFLP) analysis, 109 RubisCO-clone-inserts were subjected to sequencing and phylogenetic analysis. With the exception of a form IA RubisCO sequence cluster obtained from deep subsurface samples, which was identical to the RubisCO genes described for Ralstonia metallidurans CH34, most sequences were distantly related to a variety of RubisCO species in chemolithoautotrophic Proteobacteria. Several sequences occurred in isolated lineages. These findings suggest that autotrophic bacteria with the capability to assimilate CO2 via the Calvin Cycle pathway are widespread inhabitants of groundwater systems.

Two Pilot Plant Reactors Designed for the In Situ Bioremediation of Chlorobenzene-contaminated Ground Water: Hydrogeological and Chemical Characteristics and Bacterial Consortia

The SAFIRA in situ pilot plant in Bitterfeld, Saxonia-Anhalt, Germany, currently serves as the test site for eight different in situ approaches to remediate anoxic chlorobenzene (CB)-contaminated ground water. Two reactors, both filled with originallignite-containing aquifer material, are designed for themicrobiological in situ remediation of the ground water bythe indigenous microbial consortia. In this study, thehydrogeological, chemical and microbiological conditions of theinflowing ground water and reactor filling material are presented,in order to establish the scientific basis for the start of thebioremediation process itself. The reactors were put intooperation in June 1999. In the following, inflow CB concentrationsin the ground water varied between 22 and 33 mg L-1; achemical steady state for CB in both reactors was reachedafter 210 till 260 days operation time. The sediments werecolonized by high numbers of aerobic, iron-reducing anddenitrifying bacteria, as determined after 244 and 285 days ofoperation time. Furthermore, aerobic CB-degrading bacteria weredetected in all reactor zones. Comparative sequence analysis of16S rDNA gene clone libraries suggest the dominance of Proteobacteria (Comamonadaceae, Alcaligenaceae, Gallionella group, Acidithiobacillus) and members of theclass of low G+C gram-positive bacteria in the reactorsediments. In the inflowing ground water, sequences withphylogenetic affiliation to sulfate-reducing bacteria andsequences not affiliated with the known phyla of Bacteria, were found.

Microbial degradation of chlorobenzene under oxygen‐limited conditions leads to accumulation of 3‐chlorocatechol

Five bacterial strains (Acidovorax facilis B517, Cellulomonas turbata B529, Pseudomonas veronii B547, Pseudomonas veronii B549, and Paenibacillus polymyxa B550) isolated on chlorobenzene as the sole source of carbon and energy were screened for the accumulation of the putative metabolic intermediate 3-chlorocatechol during growth on chlorobenzene under oxygen-limited conditions in the presence and absence of nitrate (1 mM). 3-Chlorocatechol accumulated in the growth media of all five strains, but accumulation was significantly less in cultures of A. facilis B517 compared to the other four strains. The presence of nitrate did not influence the biological conversion pattern. However, biologically produced nitrite reacted with 3-chlorocatechol chemically, a reaction that masked the accumulation of 3-chlorocatechol. For P. veronii B549, a clear relationship between the presence of 3-chlorocatechol in the medium and low oxygen concentrations was demonstrated. The assumption is made that accumulation of 3-chlorocatechol is due to the low enzymatic turnover of the 3-chlorocatechol cleaving enzyme, catechol-1,2-dioxygenase, at low oxygen concentrations.

Diversity and expression of different forms of RubisCO genes in polluted groundwater under different redox conditions.

Groundwater polluted with methyl-tert-butyl ether (MTBE) and ammonium was investigated for chemolithoautotrophic CO(2) fixation capabilities based on detailed analyses of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) large subunit genes. Samples retrieved from a groundwater conditioning unit, characterized by different redox conditions, were examined for the presence of form IA, form IC (cbbL) and form II (cbbM) RubisCO genes and transcripts obtained from DNA- and RNA-extracts. Form IA RubisCO sequences, which revealed a complex and distinct variety in different sampling stations, were expressed in the original groundwater and in samples amended with oxygen, but not in the aquifer groundwater enriched with nitrate. Form IC RubisCO genes were exclusively detected in groundwater supplied with oxygen and sequences were affiliated with cbbL genes in nitrifying bacteria. cbbM genes were not expressed in the oxygen-amended groundwater, probably due to the low CO(2) /O(2) substrate specificity of this enzyme. Most form II RubisCO transcripts were affiliated with RubisCO genes of denitrifiers, which are important residents in the groundwater supplied with nitrate. The distinct distribution pattern and diversity of RubisCO genes and transcripts obtained in this study suggest that the induction of different RubisCO enzymes is highly regulated and closely linked to the actual environmental conditions.

Genetic Evidence for Bacterial Chemolithoautotrophy Based on the Reductive Tricarboxylic Acid Cycle in Groundwater Systems

Geologically and chemically distinct aquifers were screened for the presence of two genes coding for key enzymes of the reverse tricarboxylic acid (rTCA) cycle in autotrophic bacteria, 2-oxoglutarate : ferredoxin oxidoreductase (oorA) and the beta subunit of ATP citrate lyase enzymes (aclB). From 42 samples investigated, aclB genes were detected in two and oorA genes in six samples retrieved from polluted and sulfidic aquifers. aclB genes were represented by a single phylotype of almost identical sequences closely affiliated with chemolithoautotrophic Sulfurimonas species. In contrast, sequences analysis of oorA genes revealed diverse phylotypes mainly related to sequences from cultivation-independent studies.