Peter Kämpfer

Microbiological characterization of a fuel-oil contaminated site including numerical identification of heterotrophic water and soil bacteria

Seven soil samples and seven groundwater samples from a site contaminated with fuel-oil were investigated using several chemical and microbiological techniques. In soil samples, 500 to 7,500 mg/kg of total hydrocarbons were found. These samples contained no n-alkanes but iso- and branched chain alkanes. No polychlorinated biphenyls could be detected. Microbiological investigations included estimations of total cell counts, viable cell counts on different media, and numbers of methylotrophic, denitrifying, sulphate reducing, anaerobic (with the exception of methanogenic organisms), and hydrocarbon degrading bacteria. Viable and hydrocarbon degrading bacteria were found in all samples. A total of 1,366 pure cultures was characterized morphologically and physiologically and identified by numerical identification using a data base of more than 4,000 reference strains. Groundwater samples were dominated by gram-negative bacteria of the generaPseudomonas, Comamonas, Alcaligenes, andAcinetobacter, which were also found in soil samples. In addition, more grampositive bacteria belonging to the generaArthrobacter, Nocardia, andBacillus could be isolated from soil samples.

Proposed minimal standards for describing new taxa of aerobic, endospore-forming bacteria

Minimal standards for describing new taxa within the aerobic endospore-forming bacteria are proposed, following Recommendation 30b of the Bacteriological Code (1990 Revision). These minimal standards are recommended as guidelines to assist authors in the preparation of descriptions for novel taxa. They encourage broad polyphasic characterization and the construction of descriptions that are practically useful in routine diagnostic laboratories. The proposals have been endorsed by the Subcommittee on the Taxonomy of the Genus Bacillus and Related Organisms of the International Committee on Systematics of Prokaryotes.

Identification and in situ Detection of Gram-negative Filamentous Bacteria in Activated Sludge

Gram-negative filamentous bacteria are commonly observed in activated sludge and contribute to poor settlement of activated sludge flocs in secondary sedimentation tanks, a problem referred to as activated sludge bulking. However, the standard morphological identification system is of limited value for a high resolution, rapid monitoring of these bacteria. Therefore, specific 16S rRNA-targeted oligonucleotide probes were developed for Haliscomenobacter spp., Sphaerotilus spp., Leptothrix spp., Thiothrix spp., Leucothrix mucor and bacteria of the Eikelboom type 021N. Probe specificities were evaluated by nonisotopic dot blot hybridization to 145 reference strains representing a diverse collection of taxa. In situ hybridization with fluorescent probe derivatives was combined with scanning confocal laser microscopy (SCLM) for analyzing the three dimensional localization of the filaments inside the sludge flocs. Filaments could be localized even in the center of fixed flocs at a high resolution undisturbed by problems like autofluorescence.

Extensive Diversity of Ionizing-Radiation-Resistant Bacteria Recovered from Sonoran Desert Soil and Description of Nine New Species of the Genus Deinococcus Obtained from a Single Soil Sample

ABSTRACT The ionizing-radiation-resistant fractions of two soil bacterial communities were investigated by exposing an arid soil from the Sonoran Desert and a nonarid soil from a Louisiana forest to various doses of ionizing radiation using a 60Co source. The numbers of surviving bacteria decreased as the dose of gamma radiation to which the soils were exposed increased. Bacterial isolates surviving doses of 30 kGy were recovered from the Sonoran Desert soil, while no isolates were recovered from the nonarid forest soil after exposure to doses greater than 13 kGy. The phylogenetic diversities of the surviving culturable bacteria were compared for the two soils using 16S rRNA gene sequence analysis. In addition to a bacterial population that was more resistant to higher doses of ionizing radiation, the diversity of the isolates was greater in the arid soil. The taxonomic diversity of the isolates recovered was found to decrease as the level of ionizing-radiation exposure increased. Bacterial isolates of the genera Deinococcus, Geodermatophilus, and Hymenobacter were still recovered from the arid soil after exposure to doses of 17 to 30 kGy. The recovery of large numbers of extremely ionizing-radiation-resistant bacteria from an arid soil and not from a nonarid soil provides further ecological support for the hypothesis that the ionizing-radiation resistance phenotype is a consequence of the evolution of other DNA repair systems that protect cells against commonly encountered environmental stressors, such as desiccation. The diverse group of bacterial strains isolated from the arid soil sample included 60 Deinococcus strains, the characterization of which revealed nine novel species of this genus.

Brucella microti sp. nov., isolated from the common vole Microtus arvalis

Two Gram-negative, non-motile, non-spore-forming, coccoid bacteria (strains CCM 4915(T) and CCM 4916), isolated from clinical specimens of the common vole Microtus arvalis during an epizootic in the Czech Republic in 2001, were subjected to a polyphasic taxonomic study. On the basis of 16S rRNA (rrs) and recA gene sequence similarities, both isolates were allocated to the genus Brucella. Affiliation to Brucella was confirmed by DNA-DNA hybridization studies. Both strains reacted equally with Brucella M-monospecific antiserum and were lysed by the bacteriophages Tb, Wb, F1 and F25. Biochemical profiling revealed a high degree of enzyme activity and metabolic capabilities not observed in other Brucella species. The omp2a and omp2b genes of isolates CCM 4915(T) and CCM 4916 were indistinguishable. Whereas omp2a was identical to omp2a of brucellae from certain pinniped marine mammals, omp2b clustered with omp2b of terrestrial brucellae. Analysis of the bp26 gene downstream region identified strains CCM 4915(T) and CCM 4916 as Brucella of terrestrial origin. Both strains harboured five to six copies of the insertion element IS711, displaying a unique banding pattern as determined by Southern blotting. In comparative multilocus VNTR (variable-number tandem-repeat) analysis (MLVA) with 296 different genotypes, the two isolates grouped together, but formed a separate cluster within the genus Brucella. Multilocus sequence typing (MLST) analysis using nine different loci also placed the two isolates separately from other brucellae. In the IS711-based AMOS PCR, a 1900 bp fragment was generated with the Brucella ovis-specific primers, revealing that the insertion element had integrated between a putative membrane protein and cboL, encoding a methyltransferase, an integration site not observed in other brucellae. Isolates CCM 4915(T) and CCM 4916 could be clearly distinguished from all known Brucella species and their biovars by means of both their phenotypic and molecular properties, and therefore represent a novel species within the genus Brucella, for which the name Brucella microti sp. nov. with the type strain CCM 4915(T) (=BCCN 07-01(T)=CAPM 6434(T)) is proposed.

Characterization of bacterial communities from activated sludge: Culture-dependent numerical identification versus in situ identification using group- and genus-specific rRNA-targeted oligonucleotide probes

The structures of bacterial communities were studied in activated sludge samples obtained from the aerobic and anaerobic zones of a wastewater treatment plant showing enhanced phosphorous removal. Samples were analyzed by in situ hybridization with oligonucleotide probes complementary to selected regions of the 16S and 23S ribosomal RNA (rRNA) characteristic for defined phylogenetic entities (genera and larger groups). The microbial community structures revealed by molecular techniques were compared with the compositions of culturable bacterial communities, obtained from the characterization of 255 isolates from tryptone-soy (TS) agar and R2A agar. These isolates were characterized by 89 physiological tests and their cellular fatty acid patterns, and identified. Culture-dependent techniques indicated the following distribution: different Aeromonas spp. (2.7–8.3% on R2A agar; 45.0–63.7% on TS agar), Acinetobacter spp. (5.4–9.0% on R2A agar; 5.0–9.1% on TS agar), Pseudomonas spp. (up to 10% on R2A agar) and Shewanella putrefaciens (up to 3.0% on R2A agar), all members of the gamma subclass of Proteobacteria, were isolated most frequently. The relatively rare isolates of the beta subclass were identified as Acidovorax spp., Alcaligenes spp., and Comamonas spp., The Gram-positive bacteria (high DNA G+C) were assigned mainly to Arthrobacter spp., Microbacterium spp., and Mycobacterium phlei. In order to assess the in situ abundance of the most frequently isolated genus, Aeromonas, two rRNA-targeted oligonucleotide probes were developed. The two gamma proteobacterial genera Aeromonas and Acinetobacter constituted less than 5% of all bacteria. In situ, Proteobacteria belonging to the beta subclass and high G+C Gram-positive bacteria were dominant. From filamentous bacteria, Sphaerotilus spp. and Leptothrix spp. could be detected occasionally. In addition, one sample contained a high proportion of the morphologically distinct filaments of Microthrix parvicella.As for the genus Acinetobacter, the relative abundance of the most frequently gamma-proteobacterial genus Aeromonas was overestimated by the intrinsic selectivity of cultivation. Cultivation on nutrient-rich medium (TS-agar) especially supported an enhanced isolation of bacteria belonging to these two genera.

A numerical classification of the genera Streptomyces and STreptoverticillium using miniaturized physiological tests

Eight hundred and twenty-one strains of the genera Streptomyces and Streptoverticillium were physiologically characterized using a total of 329 miniaturized tests. Overall similarities of all strains were determined by numerical taxonomic techniques using the UPGMA algorithm and the SSM and the S J coefficients as measures of similarity. Test error was within acceptable limits. Comparison of photometric and visual test reading revealed overall differences of 7.45%. A total of 15 major clusters (six or more strains), 34 minor clusters (less than six strains) and 40 single-member clusters were defined at the 81.5% similarity level (S SM). Two clusters containing physiologically, and in some cases morphologically and genetically, different groups could be further subdivided at the 84.0% similarity level (S SM). Generally, similar groupings were obtained with the Jaccard coefficient at similarity levels ranging from 59.6% to 64.6% similarity (S J), with changes in the definition of clusters and subclusters. The cophenetic correlation coefficients r CS for the UPGMA/S J and the UPGMA/S sm analysis were 0.6929 and 0.8239, respectively. Several phena showed significant overlap with others, indicating the physiological variability within the species. The phenetic data in most cases confirm the major phena of the study of Williams et al. (1983), Journal of General Microbiology 129, 1743-1813 (although the cluster-groups defined in that study could only be detected in part) and the results indicate that the genus Streptomyces is still overspeciated. Some of the major groupings obtained were very much in line with chemotaxonomic and genetical data. However, several clusters containing only a few strains should be regarded as preliminary ‘species’ until further information is available. The majority of Streptoverticillium strains presently assigned to different species formed a homogeneous subcluster defined at the 84.0% similarity level (S SM). Thus, on the basis of numerical phenetic and (published) molecular genetic and chemotaxonomic data, our study supports the suggestion that members of the genus Streptoverticillium be reclassified into the genus Streptomyces.

Aerobic and facultatively anaerobic cellulolytic bacteria from the gut of the termite Zootermopsis angusticollis

Aims: To demonstrate the occurrence of cellulolytic bacteria in the termite Zootermopsis angusticollis.

Limits and possibilities of total fatty acid analysis for classification and identification of Bacillus species

Summary A numerical study of fatty acid patterns of 313 Bacillus strains was done based on standardized cultural and chemical techniques. All strains were characterized by the predominance of branched fatty acids (mainly il5:0, al5:0, often i17 :0, a17 :0, rarely i13 :0, i14 :0, i16 :0) ranging from 40% to more than 90% of total fatty acids. The ratios and proportions of the major fatty acids were found to be largely responsible for grouping strains into seven clusters (I to VII) by the UPGMA method using the correlation coefficient (S C ). Two main groups of Bacillus species can be defined on the basis of the ratio of the quantitatively predominant fatty acids i15:0 and a15:0. The species B. amyloliquefaciens , B. firmus , B. firmus-lentus intermediates, B. lentus, ‘B. freudenreichii’, B. laterosporus, B. licheniformis, B. megaterium, B. pasteurii, B. subtilis, B. alvei, B. circulans, B. coagulans, B. globisporus, B. lautus, B. macerans, B. pabuli, B. pantothenticus, B. polymyxa, B. psychrophilus and some strains of B. sphaericus (grouped into clusters I, II, and VII) are characterized by a ratio of i15 :0/a15 :0 B. thermoglucosidasius , B. badius , B. pumilus, the majority of B. sphaericus strains , B. stearothermophilus , B. cereus and B. thuringiensis grouped into clusters III to VI and exhibited a ratio of i15 :0/al5 :0 > 2. Significant quantitative variations were observed for particular fatty acids in different strains belonging to the species B. firmus , B. firmus-lentus intermediates, B. macquariensis , B. megaterium , B. subtilis , B. circulans , B. coagulans , B. macerans , and B. cereus . The species could be clustered into subgroups at a similarity level of 97.5% (S c ). With fatty acid analysis, Bacillus strains can be assigned to species groups, however, the enormous heterogeneity of fatty acid profiles within several species reflects the unsatisfactory taxonomic situation within the genus, which can be improved by further DNA-DNA hybridization studies.

Chemotaxonomic characterisation of Sphingomonas.

Based on published results and investigations done for this study, chemotaxonomic characteristics of all validly described species of the genus Sphingomonas, as well as unnamed strains of this genus and related genera such as Rhizomonas and Blastomonas, are presented. All representatives of this group, here designated as sphingomonads, contain ubiquinone (Q-10). The two different polyamine patterns characterized either by the predominant polyamine sym-homospermidine or spermidine separate the sphingomonads into two major groups. Complex polar lipid profiles were found in sphingomonads in addition to the characteristic compound sphingoglycolipid. Identical profiles were found only in a few phylogenetically highly related species. Common to all sphingomonads is a fatty acid composition with 2-hydroxy fatty acids (14:0 2OH in all currently recognized species) and the lack of 3-hydroxy acids, which distinguishes them from taxa outside this group. Qualitative and quantitative differences in the fatty acid compositions, in several cases, were also suitable for identification at the species level. Thus, the differences in the chemotaxonomic characteristics demonstrate that the analyses of these low molecular weight cell compounds are suitable for classification of sphingomonads.