Anaerobic Mineralization of Quaternary Carbon Atoms: Isolation of Denitrifying Bacteria on Dimethylmalonate

The microbial capacity to degrade simple organic compounds with quaternary carbon atoms was demonstrated by enrichment and isolation of five denitrifying strains on dimethylmalonate as the sole electron donor and carbon source. Quantitative growth experiments showed a complete mineralization of dimethylmalonate. According to phylogenetic analysis of the complete 16S rRNA genes, two strains isolated from activated sewage sludge were related to the genus Paracoccus within the α-Proteobacteria (98.0 and 98.2% 16S rRNA gene similarity to Paracoccus denitrificans^T), and three strains isolated from freshwater ditches were affiliated with the β-Proteobacteria (97.4 and 98.3% 16S rRNA gene similarity to Herbaspirillum seropedicae^T and Acidovorax facilis^T, respectively). Most-probable-number determinations for denitrifying populations in sewage sludge yielded 4.6 × 10⁴ dimethylmalonate-utilizing cells ml⁻¹, representing up to 0.4% of the total culturable nitrate-reducing population.     

Development and Application of a Real-Time PCR Approach for Quantification of Uncultured Bacteria in the Central Baltic Sea

We have developed a highly sensitive approach to assess the abundance of uncultured bacteria in water samples from the central Baltic Sea by using a noncultured member of the “Epsilonproteobacteria” related to Thiomicrospira denitrificans as an example. Environmental seawater samples and samples enriched for the target taxon provided a unique opportunity to test the approach over a broad range of abundances. The approach is based on a combination of taxon- and domain-specific real-time PCR measurements determining the relative T. denitrificans-like 16S rRNA gene and 16S rRNA abundances, as well as the determination of total cell counts and environmental RNA content. It allowed quantification of T. denitrificans-like 16S rRNA molecules or 16S rRNA genes as well as calculation of the number of ribosomes per T. denitrificans-like cell. Every real-time measurement and its specific primer system were calibrated using environmental nucleic acids obtained from the original habitat for external standardization. These standards, as well as the respective samples to be measured, were prepared from the same DNA or RNA extract. Enrichment samples could be analyzed directly, whereas environmental templates had to be preamplified with general bacterial primers before quantification. Preamplification increased the sensitivity of the assay by more than 4 orders of magnitude. Quantification of enrichments with or without a preamplification step yielded comparable results. T. denitrificans-like 16S rRNA molecules ranged from 7.1 × 10³ to 4.4 × 10⁹ copies ml⁻¹ or 0.002 to 49.7% relative abundance. T. denitrificans-like 16S rRNA genes ranged from 9.0 × 10¹ to 2.2 ×10⁶ copies ml⁻¹ or 0.01 to 49.7% relative abundance. Detection limits of this real-time-PCR approach were 20 16S rRNA molecules or 0.2 16S rRNA gene ml⁻¹. The number of ribosomes per T. denitrificans-like cell was estimated to range from 20 to 200 in seawater and reached up to 2,000 in the enrichments. The results indicate that our real-time PCR approach can be used to determine cellular and relative abundances of uncultured marine bacterial taxa and to provide information about their levels of activity in their natural environment.     

Development and Assessment of a Real-Time PCR Assay for Rapid and Sensitive Detection of a Novel Thermotolerant Bacterium, Lactobacillus thermotolerans, in Chicken Feces

A new real-time PCR assay was successfully developed using a TaqMan fluorescence probe for specific detection and enumeration of a novel bacterium, Lactobacillus thermotolerans, in chicken feces. The specific primers and probe were designed based on the L. thermotolerans 16S rRNA gene sequences, and these sequences were compared to those of all available 16S rRNA genes in the GenBank database. The assay, targeting 16S rRNA gene, was evaluated using DNA from a pure culture of L. thermotolerans, DNA from the closely related bacteria Lactobacillus mucosae DSM 13345^T and Lactobacillus fermentum JCM 1173^T, and DNA from other lactic acid bacteria in quantitative experiments. Serial dilutions of L. thermotolerans DNA were used as external standards for calibration. The minimum detection limit of this technique was 1.84 × 10³ cells/ml of an L. thermotolerans pure culture. The assay was then applied to chicken feces in two different trials. In the first trial, the cell population was 10⁴ cells/g feces on day 4 and 10⁵ cells/g feces on days 11 to 18. However, cell populations of 10⁶ to 10⁷ cells/g feces were detected in the second trial. The total bacterial count, measured by 4′,6-diamidino-2-phenylindole (DAPI) staining, was approximately 10¹¹ cells/g feces. These results suggest that in general, L. thermotolerans is a normal member of the chicken gut microbiota, although it is present at relatively low levels in the feces.     

Combined Use of Cultivation-Dependent and Cultivation-Independent Methods Indicates that Members of Most Haloarchaeal Groups in an Australian Crystallizer Pond Are Cultivable

Haloarchaea are the dominant microbial flora in hypersaline waters with near-saturating salt levels. The haloarchaeal diversity of an Australian saltern crystallizer pond was examined by use of a library of PCR-amplified 16S rRNA genes and by cultivation. High viable counts (10⁶ CFU/ml) were obtained on solid media. Long incubation times (≥8 weeks) appeared to be more important than the medium composition for maximizing viable counts and diversity. Of 66 isolates examined, all belonged to the family Halobacteriaceae, including members related to species of the genera Haloferax, Halorubrum, and Natronomonas. In addition, isolates belonging to a novel group (the ADL group), previously detected only as 16S rRNA genes in an Antarctic hypersaline lake (Deep Lake), were cultivated for the first time. The 16S rRNA gene library identified the following five main groups: Halorubrum groups 1 and 2 (49%), the SHOW (square haloarchaea of Walsby) group (33%), the ADL group (16%), and the Natronomonas group (2%). There were two significant differences between the organisms detected in cultivation and 16S rRNA sequence results. Firstly, Haloferax spp. were frequently isolated on plates (15% of all isolates) but were not detected in the 16S rRNA sequences. Control experiments indicated that a bias against Haloferax sequences in the generation of the 16S rRNA gene library was unlikely, suggesting that Haloferax spp. readily form colonies, even though they were not a dominant group. Secondly, while the 16S rRNA gene library identified the SHOW group as a major component of the microbial community, no isolates of this group were obtained. This inability to culture members of the SHOW group remains an outstanding problem in studying the ecology of hypersaline environments.     

A Broad-Host-Range, Generalized Transducing Phage (SN-T) Acquires 16S rRNA Genes from Different Genera of Bacteria

Genomic analysis has revealed heterogeneity among bacterial 16S rRNA gene sequences within a single species; yet the cause(s) remains uncertain. Generalized transducing bacteriophages have recently gained recognition for their abundance as well as their ability to affect lateral gene transfer and to harbor bacterial 16S rRNA gene sequences. Here, we demonstrate the ability of broad-host-range, generalized transducing phages to acquire 16S rRNA genes and gene sequences. Using PCR and primers specific to conserved regions of the 16S rRNA gene, we have found that generalized transducing phages (D3112, UT1, and SN-T), but not specialized transducing phages (D3), acquired entire bacterial 16S rRNA genes. Furthermore, we show that the broad-host-range, generalized transducing phage SN-T is capable of acquiring the 16S rRNA gene from two different genera: Sphaerotilus natans, the host from which SN-T was originally isolated, and Pseudomonas aeruginosa. In sequential infections, SN-T harbored only 16S rRNA gene sequences of the final host as determined by restriction fragment length polymorphism analysis. The frequency of 16S rRNA gene sequences in SN-T populations was determined to be 1 × 10⁻⁹ transductants/PFU. Our findings further implicate transduction in the horizontal transfer of 16S rRNA genes between different species or genera of bacteria.     

Molecular Fingerprint and Dominant Environmental Factors of Nitrite-Dependent Anaerobic Methane-Oxidizing Bacteria in Sediments from the Yellow River Estuary,China

Nitrite-dependent anaerobic methane oxidation (n-damo) is performed by “Candidatus Methylomirabilis oxyfera” (M. oxyfera), which connects the carbon and nitrogen global nutrient cycles. In the present study, M. oxyfera-like bacteria sequences were successfully recovered from Yellow River Estuary sediments using specific primers for 16S rRNA and pmoA genes. A M. oxyfera-like sequences analysis based on the 16S rRNA gene revealed greater diversity compared with the pmoA gene; the 16S rRNA gene sequences retrieved from the Yellow River Estuary sediments belong to groups A as well as B and were mainly found in freshwater habitats. Quantitative PCR showed that 16S rRNA gene abundance varied from 9.28±0.11×10³ to 2.10±0.13×10⁵ copies g^-1 (dry weight), and the pmoA gene abundance ranged from 8.63±0.50×10³ to 1.83±0.18×10⁵ copies g^-1 (dry weight). A correlation analysis showed that the total organic carbon (TOC) and ammonium (NH₄ ⁺) as well as the ratio of total phosphorus to total nitrogen (TP/TN) influenced the M. oxyfera-like bacteria distribution in the Yellow River Estuary sediments. These findings will aid in understanding the n-damo bacterial distribution pattern as well as their correlation with surrounding environmental factors in temperate estuarine ecosystems.     

Characterization of a Highly Enriched Dehalococcoides-Containing Culture That Grows on Vinyl Chloride and Trichloroethene

A highly enriched culture that reductively dechlorinates trichloroethene (TCE), cis-1,2-dichloroethene (cDCE), and vinyl chloride (VC) to ethene without methanogenesis is described. The Dehalococcoides strain in this enrichment culture had a yield of (5.6 ± 1.4) × 10⁸ 16S rRNA gene copies/μmol of Cl⁻ when grown on VC and hydrogen. Unlike the other VC-degrading cultures described in the literature, strains VS and BAV1, this culture maintained the ability to grow on TCE with a yield of (3.6 ± 1.3) × 10⁸ 16S rRNA gene copies/μmol of Cl⁻. The yields on an electron-equivalent basis measured for the culture grown on TCE and on VC were not significantly different, indicating that both substrates supported growth equally well. PCR followed by denaturing gradient gel electrophoresis, cloning, and phylogenetic analyses revealed that this culture contained one Dehalococcoides 16S rRNA gene sequence, designated KB-1/VC, that was identical (over 1,386 bp) to the sequences of previously described organisms FL2 and CBDB1. A second Dehalococcoides sequence found in separate KB-1 enrichment cultures maintained on cDCE, TCE, and tetrachloroethene was no longer present in the VC-H₂ enrichment culture. This second Dehalococcoides sequence was identical to that of BAV1. As neither FL2 nor CBDB1 can dechlorinate VC to ethene in a growth-related fashion, it is clear that current 16S rRNA gene-based analyses do not provide sufficient information to distinguish between metabolically diverse members of the Dehalococcoides group.     

Identification and histamine formation of Tetragenococcus isolated from Thai fermented food products

Forty-one tetrad-forming halophilic lactic acid bacteria were isolated from 7 kinds of fermented foods in Thailand. All the isolates were identified as the genus Tetragenococcus by their phenotypic characteristics. On the basis of 16S rRNA gene restriction analysis using MboI and AluI and 16S rRNA gene sequence analyses, 41 isolates could be divided into two groups (groups A and B). All 22 isolates in Group A were identified as T. halophilus. 16S rRNA gene sequences of the representative isolates, SP37-2 and KS87-1 exhibited 99.4–99.5 % similarity to that of T. halophilus ATCC 33315^T. Nineteen isolates in Group B were identified as T. muriaticus. 16S rRNA gene sequences of the representative isolates, KM1-5 and KS87-14, showed 99.0–99.6 % similarity to that of T. muriaticus JCM 10006^T. Histamine formation was determined by using HPLC and the histidine decarboxylase (hdc) gene of the newly isolated histamine-producing strain was partially sequenced. The strain KS87-14 prolifically formed histamine 10 times higher than the reported T. muriaticus JCM 10006^T. The positive detection of KS87-14 was achieved by using hdcA gene-specific primers JV16HC and JV17HC.     

Molecular cloning and characterization of ribosomal RNA genes from a blue-green alga, Anacystis nidulans

Summary Two PstI fragments (5.3x10⁶ and 4.3x10⁶ daltons) coding for Anacystis nidulans rRNA genes were cloned. The cloned rDNAs were characterized by restriction endonuclease mapping, DNA-RNA hybridization analysis and the R-loop technique. The results indicated that both fragments contained 16S, 23S and 5S rRNA genes in this order. A tRNA gene(s) was detected in the spacer region between 16S and 23S rRNA genes. The organization of A. nidulans rRNA genes resembles those of E. coli and of Euglena chloroplasts rather than those of higher plant chloroplasts.     

Verrucosispora fiedleri sp. nov., an actinomycete isolated from a fjord sediment which synthesizes proximicins

A novel filamentous actinobacterial organism, designated strain MG-37^T, was isolated from a Norwegian fjord sediment and examined using a polyphasic taxonomic approach. The organism was determined to have chemotaxonomic and morphological properties consistent with its classification in the genus Verrucosispora and formed a distinct phyletic line in the Verrucosispora 16S rRNA gene tree. It was most closely related to Verrucosispora maris DSM 45365^T (99.5 % 16S rRNA gene similarity) and Verrucosispora gifhornensis DSM 44337^T (99.4 % 16S rRNA gene similarity) but was distinguished from these strains based on low levels of DNA:DNA relatedness (~56 and ~50 %, respectively). It was readily delineated from all of the type strains of Verrucosispora species based on a combination of phenotypic properties. Isolate MG-37^T (=NCIMB 14794^T = NRRL-B-24892^T) should therefore be classified as the type strain of a novel species of Verrucosispora for which the name Verrucosispora fiedleri is proposed.     

Microcosm Enrichment of Biphenyl-Degrading Microbial Communities from Soils and Sediments

A microcosm enrichment approach was employed to isolate bacteria which are representative of long-term biphenyl-adapted microbial communities. Growth of microorganisms was stimulated by incubating soil and sediment samples from polluted and nonpolluted sites with biphenyl crystals. After 6 months, stable population densities between 8 × 10⁹ and 2 × 10¹¹ CFU/ml were established in the microcosms, and a large percentage of the organisms were able to grow on biphenyl-containing minimal medium plates. A total of 177 biphenyl-degrading strains were subsequently isolated and characterized by their ability to grow on biphenyl in liquid culture and to accumulate a yellow meta cleavage product when they were sprayed with dihydroxybiphenyl. Isolates were identified by using a polyphasic approach, including fatty acid methyl ester (FAME) analysis, 16S rRNA gene sequence comparison, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins, and genomic fingerprinting based on sequence variability in the 16S-23S ribosomal DNA intergenic spacer region. In all of the microcosms, isolates identified as Rhodococcus opacus dominated the cultivable microbial community, comprising a cluster of 137 isolates with very similar FAME profiles (Euclidean distances, <10) and identical 16S rRNA gene sequences. The R. opacus isolates from the different microcosms studied could not be distinguished from each other by any of the fingerprint methods used. In addition, three other FAME clusters were found in one or two of the microcosms analyzed; these clusters could be assigned to Alcaligenes sp., Terrabacter sp., and Bacillus thuringiensis on the basis of their FAME profiles and/or comparisons of the 16S rRNA gene sequences of representatives. Thus, the microcosm enrichments were strongly dominated by gram-positive bacteria, especially the species R. opacus, independent of the pollution history of the original sample. R. opacus, therefore, is a promising candidate for development of effective long-term inocula for polychlorinated biphenyl bioremediation.     

Quantitative Detection of the nosZ Gene, Encoding Nitrous Oxide Reductase, and Comparison of the Abundances of 16S rRNA, narG, nirK, and nosZ Genes in Soils

Nitrous oxide (N₂O) is an important greenhouse gas in the troposphere controlling ozone concentration in the stratosphere through nitric oxide production. In order to quantify bacteria capable of N₂O reduction, we developed a SYBR green quantitative real-time PCR assay targeting the nosZ gene encoding the catalytic subunit of the nitrous oxide reductase. Two independent sets of nosZ primers flanking the nosZ fragment previously used in diversity studies were designed and tested (K. Kloos, A. Mergel, C. Rösch, and H. Bothe, Aust. J. Plant Physiol. 28:991-998, 2001). The utility of these real-time PCR assays was demonstrated by quantifying the nosZ gene present in six different soils. Detection limits were between 10¹ and 10² target molecules per reaction for all assays. Sequence analysis of 128 cloned quantitative PCR products confirmed the specificity of the designed primers. The abundance of nosZ genes ranged from 10⁵ to 10⁷ target copies g⁻¹ of dry soil, whereas genes for 16S rRNA were found at 10⁸ to 10⁹ target copies g⁻¹ of dry soil. The abundance of narG and nirK genes was within the upper and lower limits of the 16S rRNA and nosZ gene copy numbers. The two sets of nosZ primers gave similar gene copy numbers for all tested soils. The maximum abundance of nosZ and nirK relative to 16S rRNA was 5 to 6%, confirming the low proportion of denitrifiers to total bacteria in soils.     

Evolution of the plastid ribosomal RNA operon in a nongreen parasitic plant: Accelerated sequence evolution,altered promoter structure,and tRNA pseudogenes

The nucleotide sequence of a 7.4 kb region containing the entire plastid ribosomal RNA operon of the nongreen parasitic plant Epifagus virginiana has been determined. Analysis of the sequence indicates that all four rRNA genes are intact and almost certainly functional. In contrast, the split genes for tRNA^Ile and tRNA^Ala present in the 16S-23S rRNA spacer region have become pseudogenes, and deletion upstream of the 16S rRNA gene has removed a tRNA^Val gene and most of the promoter region for the rRNA operon. The rate of nucleotide substitution in 16S and 23S rRNAs is several times higher in Epifagus than in tobacco, a related photosynthetic plant. Possible reasons for this, including relaxed translational constraints, are discussed.     

Sabulilitoribacter multivorans gen. nov., sp. nov., a polysaccharide-degrading bacterium of the family Flavobacteriaceae isolated from seashore sand

A Gram-negative, aerobic, non-flagellated, non-gliding and rod-shaped bacterial strain, designated M-M16^T, was isolated from seashore sand around a seaweed farm on the South Sea, South Korea, and its taxonomic position was investigated by using a polyphasic study. Strain M-M16^T grew optimally at 30 °C, at pH 7.0–8.0 and in the presence of 2 % (w/v) NaCl. Strain M-M16^T exhibited the highest 16S rRNA gene sequence similarity values to the type strains of Gaetbulibacter lutimaris (96.5 %) and Flaviramulus basaltis (95.8 %). Neighbour-joining and maximum-parsimony phylogenetic trees based on 16S rRNA gene sequences revealed that strain M-M16^T clustered with the type strains of Gaetbulibacter species and F. basaltis. Strain M-M16^T contained MK-6 as the predominant menaquinone and iso-C_15:1 G, iso-C_15:0 and iso-C_17:0 3-OH as the major fatty acids. The major polar lipids detected in strain M-M16^T were phosphatidylethanolamine and one unidentified lipid. The DNA G+C content of strain M-M16^T was 37.4 mol%. The phylogenetic and chemotaxonomic data and other phenotypic properties revealed that strain M-M16^T represents a novel genus and species within the family Flavobacteriaceae, for which the name Sabulilitoribacter multivorans gen. nov., sp. nov. is proposed. The type strain of S. multivorans is M-M16^T (= KCTC 32326^T = CCUG 63831^T).     

Comparison of 16S rRNA gene phylogeny and functional tfdA gene distribution in thirty-one different 2,4-dichlorophenoxyacetic acid and 4-chloro-2-methylphenoxyacetic acid degraders

31 different bacterial strains isolated using the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole source of carbon, were investigated for their ability to mineralize 2,4-D and the related herbicide 4-chloro-2-methylphenoxyacetic acid (MCPA). Most of the strains mineralize 2,4-D considerably faster than MCPA. Three novel primer sets were developed enabling amplification of full-length coding sequences (CDS) of the three known tfdA gene classes known to be involved in phenoxy acid degradation. 16S rRNA genes were also sequenced; and in order to investigate possible linkage between tfdA gene classes and bacterial species, tfdA and 16S rRNA gene phylogeny was compared. Three distinctly different classes of tfdA genes were observed, with class I tfdA sequences further partitioned into the two sub-classes I-a and I-b based on more subtle differences. Comparison of phylogenies derived from 16S rRNA gene sequences and tfdA gene sequences revealed that most class II tfdA genes were encoded by Burkholderia sp., while class I-a, I-b and III genes were found in a more diverse array of bacteria.     

Bacterial community compositions of tomato (Lycopersicum esculentum Mill.) seeds and plant growth promoting activity of ACC deaminase producing Bacillus subtilis (HYT-12-1) on tomato seedlings

Study of endophytic bacteria within plant seeds is very essential and meaningful on account of their heritability and versatility. This study investigated Bacillus bacterial communities within the seeds of four commercial tomato varieties, by 16S rRNA gene PCR-RFLP (restriction fragment length polymorphism). Phylogenetic analysis of 16S rRNA gene sequences indicated that the 22 representative isolates belonged to five species of genus Bacillus and the bacterial compositions showed remarkable differences among tomato varieties. Isolates exhibited multiple plant growth promoting (PGP) traits: 37 % of indole-3-acetic acid production; 37 % of phosphate solubilization; 24 % of siderophores production; 85 % of potential nitrogen fixation and 6 % of 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity. Isolate HYT-12-1 was shown to have highest ACC deaminase activity (112.02 nmol α-ketobutyrate mg^?1 protein h^?1) among the five ACC deamiase producing strains. 16S rRNA gene sequencing indicated that the isolate HYT-12-1 shared the highest sequence similarity (100 %) with B. subtilis. PGP experiments under gnotobiotic and greenhouse conditions revealed the ability of strain HYT-12-1 to enhance the growth of tomato seedlings. This is the first study to describe endophytic Bacillus communities within tomato seeds, and the results suggest that B. subtilis strain HYT-12-1 would have a great potential for industrial application as biofertilizer in the future.     

Detection of Jeotgalicoccus spp. in poultry house air

Investigations of bioaerosols collected from turkey, chicken and duck houses, as well as from a duck slaughterhouse, each in triplicate, revealed that 4–18% of 16S rRNA gene sequences in investigated 16S rRNA gene clone libraries were closely related to Jeotgalicoccus spp. J. halotolerans- and J. psychrophilus-related sequences were obtained in all investigated bioaerosol samples and formed a distinct group with sequences of both species type strains, which were collectively entitled Jeot-cluster-I. For a quantification of Jeot-cluster-I bacteria, a group specific PCR primer combination targeting the 16S rRNA genes was developed. Estimated concentrations by quantitative real-time PCR analyses revealed cell numbers between 10⁴ and 10⁶ Jeotgalicoccus cells m⁻³ air in turkey, duck, and chicken houses, respectively. These results indicated the remarkable proportion (1–39%) of total cell counts and the hitherto unknown wide distribution of Jeotgalicoccus spp. in the poultry rearing industry.     

Quantification of Hyphomicrobium Populations in Activated Sludge from an Industrial Wastewater Treatment System as Determined by 16S rRNA Analysis

The bacterial community structure of the activated sludge from a 25 million-gal-per-day industrial wastewater treatment plant was investigated using rRNA analysis. 16S ribosomal DNA (rDNA) libraries were created from three sludge samples taken on different dates. Partial rRNA gene sequences were obtained for 46 rDNA clones, and nearly complete 16S rRNA sequences were obtained for 18 clones. Seventeen of these clones were members of the beta subdivision, and their sequences showed high homology to sequences of known bacterial species as well as published 16S rDNA sequences from other activated sludge sources. Sixteen clones belonged to the alpha subdivision, 7 of which showed similarity to Hyphomicrobium species. This cluster was chosen for further studies due to earlier work on Hyphomicrobium sp. strain M3 isolated from this treatment plant. A nearly full-length 16S rDNA sequence was obtained from Hyphomicrobium sp. strain M3. Phylogenetic analysis revealed that Hyphomicrobium sp. strain M3 was 99% similar to Hyphomicrobium denitrificans DSM 1869^T in Hyphomicrobium cluster II. Three of the cloned sequences from the activated sludge samples also grouped with those of Hyphomicrobium cluster II, with a 96% sequence similarity to that of Hyphomicrobium sp. strain M3. The other four cloned sequences from the activated sludge sample were more closely related to those of the Hyphomicrobium cluster I organisms (95 to 97% similarity). Whole-cell fluorescence hybridization of microorganisms in the activated sludge with genus-specific Hyphomicrobium probe S-G-Hypho-1241-a-A-19 enhanced the visualization of Hyphomicrobium and revealed that Hyphomicrobium appears to be abundant both on the outside of flocs and within the floc structure. Dot blot hybridization of activated sludge samples from 1995 with probes designed for Hyphomicrobium cluster I and Hyphomicrobium cluster II indicated that Hyphomicrobium cluster II-positive 16S rRNA dominated over Hyphomicrobium cluster I-positive 16S rRNA by 3- to 12-fold. Hyphomicrobium 16S rRNA comprised approximately 5% of the 16S rRNA in the activated sludge.     

Fermentation of ginseng extracts by Penicillium simplicissimum GS33 and anti-ovarian cancer activity of fermented products

A total of 58 isolates of β-glucosidase-producing microorganisms were isolated from soil around the wild ginseng roots under forest using Esculin-R2A agar. Among these isolates, strain GS33 showed a strong ability to convert ginsenosides Rb1, Rb2, Rc, and Rd into F2, Rg3, C-K, and convert ginsenoside Rg1 into Rh1, and F1. Fermented ginseng products can inhibit ES-2 cells growth and the IC₅₀ value was 0.73 mg ml^?1. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the strain GS33 belongs to the genus Penicillium and is most closely related to Penicillium simplicissimum (99 %).