Anna Rusznyák

Diversity of Alkaliphilic and Alkalitolerant Bacteria Cultivated from Decomposing Reed Rhizomes in a Hungarian Soda Lake

Bacterial communities associated with decomposing rhizomes of Phragmites australis were investigated in Lake Fertő (Neusiedlersee, Hungary). Alkaliphilic and alkalitolerant strains were isolated on cellulose-containing alkaline medium spread with dilutions of scrapings taken from the surface of the decaying plant material. Fifty-one strains were grouped by numerical analysis based on physiological tests and BIOLOG sole carbon source utilization data. The strains identified by 16S rDNA sequence comparisons included members of low G+C Gram positives (Marinibacillus marinus, Bacillus cereus, and Exiguobacterium aurantiacum), high G+C Gram positives (Nesterenkonia halobia and Dietzia natronolimnea), α-proteobacteria (Pannonibacter phragmitetus), and γ-proteobacteria (Pseudomonas pseudoalcaligenes and Halomonas venusta). Most of the strains were characterized by aerobic chemoorganotrophic respiratory metabolism and utilized several different carbon sources, although no direct cellulolytic activity was observed. Results of the pH and salt tolerance tests revealed optimuma in most cases at pH 11 and at the presence of 2.5–5% NaCl. These bacteria probably occupy niches in the aerobic, alkaline, water-influenced environments on the decomposing reed surfaces.

Diversity of Sulfate-Reducing Bacteria Inhabiting the Rhizosphere of Phragmites australis in Lake Velencei (Hungary) Revealed by a Combined Cultivation-based and Molecular approach

The community structure of sulfate-reducing bacteria (SRB) associated with reed (Phragmites australis) rhizosphere in Lake Velencei (Hungary) was investigated by using cultivation-based and molecular methods. The cultivation methods were restricted to recover lactate-utilizing species with the exclusion of Desulfobacter and some Desulfobacterium species presumably not being dominant members of the examined community. The most-probable-number (MPN) estimations of lactate-utilizing SRB showed that the cell counts in reed rhizosphere were at least one order of magnitude higher than that in the bulk sediment. The number of endospores was low compared to the total SRB counts. From the highest positive dilution of MPN series, 47 strains were isolated and grouped by restriction fragment length polymorphism (RFLP) analysis of the amplified 16S ribosomal RNA (rRNA) and dsrAB (dissimilatory sulfite reductase) genes. Contrary to the physiological diversity of the isolates, the combined results of RFLP analysis revealed higher diversity at species as well as at subspecies level. Based on the partial 16S rRNA sequences, the representative strains were closely affiliated with the genera Desulfovibrio and Desulfotomaculum. The partial dsrAB sequences of the clones, recovered after isolation and PCR amplification of the community DNA, were related to hitherto uncultured species of the genera Desulfovibrio and Desulfobulbus. Nevertheless, the representative of the second largest clone group was shown to be closely affiliated with the sequenced dsrAB gene of a strain isolated from the same environment and identified as Desulfovibrio alcoholivorans. Another clone sequence was closely related to a possible novel species also isolated within the scope of this work.

Algological and bacteriological investigations on reed periphyton in Lake Velencei, Hungary

In the shallow Lake Velencei (surface area 24.5 km2) reed communities (Scirpo-Phragmitetum) are of great significance due to extensive metabolic activity of algal-bacterial associations developing on their submerged surfaces. Samples for algological and bacteriological studies were taken in April 2000 and July 2001 at five sites of the lake and were analysed with traditional methods as well as with tools of molecular biology (16S rDNA sequencing). These investigations have shown that (i) The original mosaic structure of the lake disappeared; periphytic algae became spatially uniform both in terms of taxonomic composition and abundance. (ii) The biodiversity of the reed periphyton decreased. The dominance of the weed algal species Achnanthes minutissima Kütz. increased significantly. (iii) The most frequent bacteria were members of the genera Pseudomonas, Acinetobacter, Shewanella and Agrobacterium characterised by oxidative chemoorganotrophic heterotrophic- and Aeromonas and Bacillus species with fermentative metabolism. (iv) Among the studied bacterial strains both plant growth promoting bacteria, members of Pseudomonas fluorescens group, and potential plant pathogens (Agrobacterium, Aureobacterium, Curtobacterium) were present.

Seasonal and spatial variability of sediment bacterial communities inhabiting the large shallow Lake Balaton

Seasonal studies of surface sediment bacterial communities, from two basins with differing trophic states within Lake Balaton (Hungary), were carried out using molecular (denaturing gradient gel electrophoresis, DGGE) and cultivation-based techniques. The presence of polyphosphate accumulates was tested using Neisser staining, and phosphatase activity was investigated on organic phosphorus (P) compound. Aerobic viable cell counts were significantly higher in the eutrophic than mesotrophic basin in each season. The lowest viable counts were observed in the autumn and the highest in spring and summer month in both basins. The DGGE fingerprints of the samples reflected that the composition of sediment bacterial communities in the two basins were distinct in spring and summer, and similar in autumn, but similarly diverse in all seasons. On the basis of partial 16S rDNA sequences, the 216 strains were affiliated with six major bacterial lineages: Firmicutes; Actinobacteria, Bacteroidetes, Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria. Common species characterized from both basins constituted up to 66% of all identified phylotypes. Strains related to Bacillus sp. were dominant in all but one sample. Isolates affiliated with Aeromonas sp. prevailed in the sample taken from the mesotrophic basin in spring. The majority of the strains showed excess poly-P accumulation. Association of Neisser staining and phosphatase activity test results suggested that excess poly-P accumulation serves as P storage for sediment bacteria. Our study implied the importance of Firmicutes, Actinobacteria, Alphaproteobacteria, and Aeromonas species in benthic bacterial P retention.

Metabolic Activity and Phylogenetic Diversity of Reed (Phragmites australis) Periphyton Bacterial Communities in a Hungarian Shallow Soda Lake

In the present study, the species composition and potential metabolic activities of bacterial communities of reed Phragmites australis (Cav.) (Trin. ex Steudel) periphyton from Lake Velencei were studied by cultivation-based and metabolic fingerprinting methods. Serially diluted spring biofilm samples were used to test the community-level physiological profiling (CLPP) using BIOLOG microplates, and for plating onto different media. On the basis of their morphological, biochemical, and physiological test results, 173 strains were clustered by numerical analysis. Representatives of amplified ribosomal DNA restriction analysis (ARDRA) groups were identified by their 16S rDNA sequence comparison. Based on the results of the CLPP investigations, regional differences were detected among the utilized substrate numbers and types, parallel with the increase in incubation time. The phenotypic test results of the strains showed considerable variability with respect to the sampling sites and the media used for cultivation. The most frequently isolated strains were identified as members of genera Agrobacterium, Pseudomonas (P. anguilliseptica, P. marginalis, P. alcaligenes, P. fragi) with aerobic or facultative anaerobic respiratory metabolism, and the species Aeromonassobria and A. veronii with strong facultative fermentative metabolism. Other strains were identified as Gram-positive Arthrobacter, Bacillus, and Kocuria species. The rarely isolated strains were members of β-Proteobacteria (Acidovorax, Delftia, Hydrogenophaga, and Rhodoferax), γ-Proteobacteria (Psychrobacter and Shewanella), low G + C Gram-positives (Brevibacillus, Paenibacillus, and Exiguobacterium) and high G + C Gram-positives (Aureobacterium and Microbacterium).

Bacillus alkalisediminis sp. nov., an alkaliphilic and moderately halophilic bacterium isolated from sediment of extremely shallow soda ponds

Alkaliphilic strains characterized by optimal growth at pH 9.0 and 5 % (w/v) NaCl designated K1-25(T) and H3-93 were isolated from extremely shallow soda ponds located in Hungary. Cells of both strains were Gram-stain-positive, non-motile, straight rods and formed central, ellipsoidal endospores with swollen sporangia. The isolates were aerobic, catalase-positive, oxidase-negative and contained a peptidoglycan of type A1γ based on meso-diaminopimelic acid. In both strains, menaquinone-7 (MK-7) was the predominant isoprenoid quinone and the major cellular fatty acids were anteiso-C(15 : 0) and iso-C(15 : 0). The DNA G+C contents of strains K1-25(T) and H3-93 were 39.0 and 36.3 mol%, respectively. 16S rRNA gene sequence-based phylogenetic analysis revealed 99.2 % similarity between strains K1-25(T) and H3-93 and the novel isolates had the highest similarities to Bacillus akibai 1139(T) (97.8 and 98.3 %, respectively), Bacillus wakoensis N-1(T) (97.0 and 97.4 %), Bacillus okhensis Kh10-101(T) (97.1 and 97.4 %) and Bacillus krulwichiae AM31D(T) (96.9 and 97.1 %). DNA-DNA hybridization between our strains and the type strains of closely related Bacillus species was lower than 70 %. Although DNA-DNA hybridization between strains K1-25(T) and H3-93 was 27 %, the phenotypic and chemotaxonomic data did not support the differentiation of these two strains into separate species. Therefore, they represent genomovars of a novel species, for which the name Bacillus alkalisediminis sp. nov. is proposed. The type strain is K1-25(T) ( = DSM 21670(T)  = NCAIM B02301(T)).

Cellulomonas phragmiteti sp. nov., a cellulolytic bacterium isolated from reed (Phragmites australis) periphyton in a shallow soda pond

An alkalitolerant and moderately halophilic strain, designated KB23(T), characterized by optimal growth at pH 8.0-9.0 and in the presence of 5-7 % (w/v) NaCl, was isolated from a reed (Phragmites australis) periphyton sample originating from an extremely shallow, alkaline soda pond located in Hungary. Cells of strain KB23(T) were Gram-stain-positive, motile straight rods. Strain KB23(T) was facultatively anaerobic, catalase-positive, oxidase-negative and contained peptidoglycan type A4β (L-Orn-D-Asp). MK-9(H4) was the predominant isoprenoid quinone and anteiso-C(15 : 0), C(16 : 0) and anteiso-C(15 : 1) were the major cellular fatty acids. The DNA G+C content of strain KB23(T) was 74.8 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that this strain belongs to the genus Cellulomonas and that it is related most closely to Cellulomonas flavigena DSM 20109(T) (97.35 % similarity), Cellulomonas terrae DB5(T) (96.81 %), Cellulomonas iranensis O(T) (96.75), Cellulomonas chitinilytica X.bu-b(T) (96.60 %), Cellulomonas persica I(T) (96.53 %), Cellulomonas composti TR7-06(T) (96.45 %), Cellulomonas biazotea DSM 20112(T) (96.34 %) and Cellulomonas fimi DSM 20113(T) (96.20 %). According to these results, together with DNA-DNA hybridization and physiological data, strain KB23(T) is considered to represent a novel species of the genus Cellulomonas, for which the name Cellulomonas phragmiteti sp. nov. is proposed. The type strain is KB23(T) ( = DSM 22512(T)  = NCAIM B002303(T)).

Phylogenetic and metabolic bacterial diversity of Phragmites australis periphyton communities in two Hungarian soda ponds

Bacterial diversity of reed (Phragmites australis) periphyton communities of Kelemen-szék and Nagy-Vadas (two Hungarian soda ponds) was investigated using molecular cloning and cultivation-based techniques. The majority of the 80 Kelemen-szék and 72 Nagy-Vadas bacterial isolates proved to be moderately halophilic and alkaliphilic. A great proportion of the isolates showed phosphatase and urease activity, utilized aesculin, citrate and certain biopolymers (e.g., gelatine and tween 80). Partial 16S rDNA sequence analysis of 33 Kelemen-szék and 20 Nagy-Vadas ARDRA group representatives showed Gram-positive (Nesterenkonia, Cellulomonas, Dietzia, Bacillus and Planococcus) dominance at both sampling sites. Species of the genera Acidovorax, Hydrogenophaga (β-Proteobacteria) and Flavobacterium, Sphingobacterium (Bacteroidetes) were represented only from Kelemen-szék. Altogether 16 isolates showed low sequence similarity with yet described bacteria and may represent novel taxa. Screening of the 16S rRNA gene libraries of 129 Kelemen-szék and 158 Nagy-Vadas clones resulted in 30 and 28 different ARDRA groups, respectively. Sequence analysis revealed a Gram-negative (Rheinheimera, Aquimonas, Cellvibrio, Flavobacterium and Sphingobacterium) dominated phylogenetic diversity. A high number of the clones were affiliated with uncultured bacterial clones described from diverse environmental samples.