rDNA Clone Research Articles

In situ diets of the bloom-forming dinoflagellate Noctiluca scintillans in Daya Bay

Red Noctiluca scintillans is a common heterotrophic dinoflagellate that forms blooms in temperate, subtropical, and tropical coastal ecosystems. The diet of this species plays an important role in its cell growth, development, and reproduction. Because limited gene diversity data are available regarding prey of this species, its diet in Daya Bay during a boreal winter bloom is reported using an integrated approach involving light microscopy, single cell isolation and plastid 16S rDNA cloning, and 18S rDNA V4 and V9 region amplification using isolated cells and environmental DNA as templates with high-throughput sequencing. While conventional light microscopy reveals the diet of this species to comprise Coscinodiscus sp. and Stephanopyxis turris (diatoms), copepod eggs, and detritus, plastid gene diversity identifies a diet comprising diatoms, cyanobacteria, and bacteria, and 18S rDNA high-throughput sequencing reveals a diet comprising 36 eukaryote families (primarily copepods, as well as diatoms, dinoflagellates, Ochrophyta, Haptophytes, Chordata, Cercozoans, Chlorophyta, Polychaeta, and ciliates). Dietary staples include copepods, diatoms, dinoflagellates, Ochrophyta, and Synechococcus. High copepod abundance in prey may reflect their relatively high abundance in environmental seawater. Thus, N. scintillans is generally omnivorous but prefers dominant phytoplankton taxa, including Rhizosoleniaceae, Leptocylindraceae, and Cymatosiraceae (diatoms), as well as Gonyaulacaceae (dinoflagellates). An integrated multi-disciplinary approach provides a more comprehensive picture of N. scintillans diet in Daya Bay, and an improved understanding of this species’ ecological niche and trophic role in marine ecosystems.

Dynamic distribution of Massilia spp. in sewage, substrate, plant rhizosphere/phyllosphere and air of constructed wetland ecosystem.

Massilia bacteria are widely distributed and have various ecological functions. Preliminary studies have shown that Massilia is the dominant species in constructed wetland ecosystems, but its species composition and distribution in constructed wetlands are still unclear. In this paper, the in-house-designed primers were used to construct a 16S rDNA clone library of Massilia. The RFLP sequence analysis method was used to analyze the diversity of Massilia clone library and the composition of Massilia in sewage, substrate, plant rhizosphere, plant phyllosphere and air in a constructed wetland sewage treatment system. Redundancy analysis (RDA) and canonical correspondence analysis (CCA) were used to analyze the correlation between environmental factors and the population characteristics of Massilia in the corresponding environment. The dominant species of Massilia were analyzed for differences. The results showed that the 16S rDNA clone library in primer 5 worked well. According to the clone library diversity index analysis, the richness of Massilia varied significantly in different environments in different seasons, where the overall summer and autumn richness was higher than that in the spring and winter. The relative abundance of 5 Massilia in the constructed wetland ecosystem was greater than 1% in all samples, which were M. alkalitolerans, M. albidiflava, M. aurea, M. brevitalea, and M. timonae. The seasonal variation of dominant genera was significantly correlated with environmental factors in constructed wetlands. The above results indicated that the species of Massilia were abundant and widely distributed in the constructed wetland ecosystem, and there were significant seasonal differences. In addition, the Massilia clone library of constructed wetland was constructed for the first time in this study and the valuable data of Massilia community structure were provided, which was conducive to the further study of microbial community in constructed wetland.

The Dynamic Shift of Bacterial Communities in Hybrid Anaerobic Baffled Reactor (ABR)—Aerobic Granules Process for Berberine Pharmaceutical Wastewater Treatment

Because of its anticancer, anti-inflammatory, and antibiotic properties, berberine has been used extensively in medication. The extensive production of berberine results in the generation of wastewater containing concentrated residual berberine. However, to date, limited related studies on the biological treatment of berberine wastewaters have been carried out. A lab-scale anaerobic baffled reactor (ABR)–aerobic granular sludge (AGS) process was developed for berberine removal from synthetic wastewater. The system showed effective removal of the berberine. In order to better understand the roles of the bacterial community, the ABR–aerobic granular sludge system was operated in the state with the highest BBR removal rate in this study. The bacterial community dynamics were studied using the 16S rDNA clone library. The results showed that the hybrid ABR-AGS process achieved 92.2% and 94.8% overall removals of berberine and COD, respectively. Bacterium was dominant species in ABR, while the CFB group bacteria and Betaproteobacteria were dominant species in AGS process. The uncultured bacterium clone B135, Bacillus endophyticus strain a125, uncultured bacterium mle1-42, uncultured bacterium clone OP10D15, and uncultured bacterium clone B21.29F54 in ABR, and uncultured bacterium clone F54, uncultured bacterium clone ZBAF1-105, uncultured bacterium clone SS-9, and uncultured bacterium clone B13 in AGS process were identified as functional species in the biodegradation of berberine and/or its metabolites. Both anaerobic and aerobic bacterial communities could adapt appropriately to different berberine selection pressures because the functional species’ identical functions ensured comparable pollutant removal performances. The information provided in this study may help with future research in gaining a better understanding of berberine biodegradation.

The Relationship Between Dust Sources and Airborne Bacteria in the Southwest of Iran.

The biological agents are carried from deserts and dried lands to long distances by high dust volumes. Their adverse effects can be reduced by specifying and controlling dust sources and their related biological agents. Thus, the current work examined the relationship between the bacteria in air and soil samples by taking samples from the soil surface of two dust sources, as well as from air samples during spring from Khorramshahr and Abadan cities. The dust event is the most influential factor on airborne bacteria. There is an insignificant negative (-0.06), insignificant positive (0.14), and weak positive (0.24) correlation between airborne bacteria and UV radiation, relative humidity, and temperature, respectively. After preparing a 16S ribosomal DNA (rDNA) clone library from the soil and air samples, operational taxonomic unit picking and taxonomic assignment were conducted using QIIME Virtual Box. In the present work, Bacillus was the dominant species. The relationship between dust sources and air samples was determined by principal component analysis. Bacteria in the Hoor-Al-Azim dust source and airborne bacteria on dusty and non-dusty days showed a more significant correlation compared to bacteria in the Shadegan dust source. Source Tracker software was used to estimate the contribution of dust sources. The primary source of dust was associated with the dried areas of Hoor-Al-Azim on the non-dusty and dusty days. Finally, the long transport of airborne bacteria was assessed by moderate resolution imaging spectroradiometer (MODIS) and the back trajectory model of Hybrid Single-Particle Lagrangian Integrated Trajectory (HYSPLIT) data. The research findings can help decision-makers prioritize dust sources to control the adverse effects of dust.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11356-022-21563-6.

Bacteroides fragilis Supplementation Deteriorated Metabolic Dysfunction, Inflammation, and Aorta Atherosclerosis by Inducing Gut Microbiota Dysbiosis in Animal Model.

Background: The gut microbial ecosystem is an important factor that regulates host health and the onset of chronic diseases, such as inflammatory bowel diseases, obesity, hyperlipidemia, and diabetes mellitus, which are important risk factors for atherosclerosis. However, the links among diet, microbiota composition, and atherosclerotic progression are unclear. Methods and results: Four-week-old mice (-/- mice, C57Bl/6) were randomly divided into two groups, namely, supplementation with culture medium (control, CTR) and Bacteroides fragilis (BFS), and were fed a high-fat diet. The gut microbiota abundance in feces was evaluated using the 16S rDNA cloning library construction, sequencing, and bioinformatics analysis. The atherosclerotic lesion was estimated using Oil Red O staining. Levels of CD36, a scavenger receptor implicated in atherosclerosis, and F4/80, a macrophage marker in small intestine, were quantified by quantitative real-time PCR. Compared with the CTR group, the BFS group showed increased food intake, fasting blood glucose level, body weight, low-density lipoprotein level, and aortic atherosclerotic lesions. BFS dramatically reduced Lactobacillaceae (LAC) abundance and increased Desulfovibrionaceae (DSV) abundance. The mRNA expression levels of CD36 and F4/80 in small intestine and aorta tissue in the BFS group were significantly higher than those in the CTR group. Conclusions: gut microbiota dysbiosis was induced by BFS. It was characterized by reduced LAC and increased DSV abundance and led to the deterioration of glucose/lipid metabolic dysfunction and inflammatory response, which likely promoted aorta plaque formation and the progression of atherosclerosis.

BACTERIOME ASSOCIATED WITH Rhizophora mangle SEDIMENTS WITHIN BRAZIL SEMI-ARID MANGROVES

Microorganisms play important roles in nutrient cycling in mangrove ecosystems and knowledge on the plant/microorganism association is essential to better understand the functioning of this environment. Rhizophora mangle is the dominant tree species within Brazilian mangroves and little information is available on the microbiota associated with this plant species. In this context, the aim of this study was to survey the taxonomic diversity of bacteria in the R. mangle root zones in mangroves within the semi-arid region of Northeast Brazil submitted to different human disturbances, intending to determine the bacterialfingerprint associated with this habitat. The total DNA extracted from sediments of different mangroves was pooled and used for construction of 16S rDNA cloning libraries, which resulted in 663 sequences with an average size of 809 bp. All mangroves were rich in different phyla of the Bacteria domain, with Acidobacteria, Bacteroidetes, Chloroflexi, and Proteobacteria being detected in all locations. Proteobacteria was dominant in all mangroves, and it was mainly represented by Alpha, Delta, and Gammaproteobacteria. The greatest richness was found in the Timonha river mangrove, with 13 phyla, a location considered more preserved compared to other mangroves. The lowest richness was found in Ceará river mangrove, with only seven phyla. This mangrove is threatened by intense urbanization. The results clearly showed that the taxonomic diversity of bacteria from mangroves subjected to intense urbanization have decreased, highlighting the risks of these changes for the functioning of important microbe-mediated processes and related ecosystem services.
 Keywords: brazilian mangroves, semi-arid, Rhizophora mangle, rRNA 16S, clone library.

The Ribosomal DNA Loci of the Ancient Monocot Pistia stratiotes L. (Araceae) Contain Different Variants of the 35S and 5S Ribosomal RNA Gene Units.

The freshwater plant water lettuce (Pistia stratiotes L.) grows in warm climatic zones and is used for phytoremediation and biomass production. P. stratiotes belongs to the Araceae, an ecologically and structurally diverse early monocot family, but the phylogenetic relationships among Araceae members are poorly understood. Ribosomal DNAs (rDNAs), including the 35S and 5S rDNA, encode the RNA components of ribosomes and are widely used in phylogenetic and evolutionary studies of various plant taxa. Here, we comprehensively characterized the chromosomal locations and molecular organization of 35S and 5S rDNA genes in water lettuce using karyological and molecular methods. Fluorescence in situ hybridization revealed a single location for the 35S and 5S rDNA loci, each on a different pair of the species’ 28 chromosomes. Molecular cloning and nucleotide sequencing of 35S rDNA of P. stratiotes, the first representative Araceae sensu stricto in which such a study was performed, displayed typical structural characteristics. The full-length repeat showed high sequence conservation of the regions producing the 18S, 5.8S, and 25S rRNAs and divergence of the internal transcribed spacers ITS1 and ITS2 as well as the large intergenic spacer (IGS). Alignments of the deduced sequence of 18S rDNA with the sequences available for other Araceae and representatives of other clades were used for phylogenetic analysis. Examination of 11 IGS sequences revealed significant intra-genomic length variability due to variation in subrepeat number, with four types of units detected within the 35S rDNA locus of the P. stratiotes genome (estimated size 407 Mb/1C). Similarly, the 5S rDNA locus harbors gene units comprising a conserved 119-bp sequence encoding 5S rRNA and two types of non-transcribed spacer (NTS) sequences. Type I was classified into four subtypes, which apparently originated via progressive loss of subrepeats within the duplicated NTS region containing the 3’ part of the 5S rRNA gene. The minor Type II NTS is shorter than Type I and differs in nucleotide composition. Some DNA clones containing two or three consecutive 5S rDNA repeats harbored 5S rDNA genes with different types of NTSs, confirming the mosaic composition of the 5S rDNA locus.

Physiological Response of an Oil-Producing Microalgal Strain to Salinity and Light Stress.

By separating and extracting algae from the collected water samples, an oil-producing diatom strain was obtained. Microscopic observation of the strain revealed that its morphological characteristics were highly similar to those of the genus Cyclotella. The cloning of 18S rDNA and phylogenetic analysis showed that the algae were clustered with Cyclotella menegheniana with a high support rate, indicating that the alga was C. menegheniana. The fatty acid content of the alga was determined and found to be mainly C14, C16, and C18 fatty acids, which were in accordance with the relevant standards for edible oil. In this study, different gradient levels of salinity and light were set to investigate the culture and bioactive substance production of C. menegheniana. The results showed that the best growth condition was achieved when the salinity was 15 g·L−1, and its biomass and oil content were the highest at 0.27 g·L−1 and 21%, respectively. The final biomass was the highest when the light intensity was 2000 Lux and the oil content was 18.7%. The results of the study provided a basis for the large-scale production of edible oils and biodiesel.

Особенности микробиоты у пациентов с болезнью Крона в зависимости от наличия синдрома избыточного бактериального роста

Aim: to evaluate microbiota in Crohn’s disease depending on the presence/absence of small intestinal bacterial overgrowth (SIBO). Patients and Methods: thirteen patients with Crohn’s disease affecting the small intestine were enrolled. Gut microbiota composition was analyzed using the 16S rRNA gene sequencing. 16S rDNA clone libraries were generated similarly. All patients underwent lactulose hydrogen breath test using Gastroplus+ monitor to establish SIBO. A positive lactulose hydrogen breath test indicated SIBO. Results: hydrogen breath test revealed SIBO in 8 patients with Crohn’s disease affecting the small intestine (61.5%). All these patients were women (mean age 40.5 [34.5; 51.2] years). In 5 patients (mean age 32.4 [28.7; 36.5] years), SIBO was not detected. The metagenome analysis of intestinal bacteria identified certain significant differences in microbiota composition in patients with Crohn’s disease depending on the presence/absence of SIBO. In Crohn’s disease affecting the small intestine, SIBO was associated with greater occurrence of Chryseobacterium spp. (р=0.045), Klebsiella spp. (р=0.045), Phocaecola operational taxonomic unit (OTU) (р=0.045), Enterobacteriaceae OTU (р=0.045), Pasteurellales. (р=0.031), Peptoniphilaceae (р=0,023), Solobacterium spp. (р=0.002), Haemophilus spp. (р=0.031), Lachnospiracea OTU (р=0.045), and Bifidobacterium spp. (р=0.019). Conclusions: a taxonomic shift with the reduction of Firmicutes and Bifidobacterium and increase of Proteobacteria (i.e., Klebsiella spp. and Enterobacteriaceae OTU) was revealed in patients with Crohn’s disease and SIBO. KEYWORDS: inflammatory bowel disease, Crohn’s disease, microbiota, small intestinal bacterial overgrowth, hydrogen respiratory test. FOR CITATION: Kulygina Yu.A., Osipenko M.F., Alikina T.Yu. Microbiota pattern in Crohn’s disease depending on the presence ofsmall intestinal bacterial overgrowth. Russian Medical Inquiry. 2022;6(5):221–226 (in Russ.). DOI: 10.32364/2587-6821-2022-6-5-221-226.

Organization and polymorphysm of 5S rDNA intergenic spacer of blackthorn (Prunus spinosa L.)

Aim. The 5S rDNA repeated units consist of conserved regions encoding 5S rRNA and variable intergenic spacers (IGS). The IGS sequences are commonly used as molecular markers for low-ranking phylogenetic, phylogeographical and microevolutionary studies. However, this genomic region still remains undescribed for the waist majority of genera in the Rosaceae family. Here we present the first report of the IGS molecular organization and polymorphism for the widespread member of the Rosaceae family, Prunus spinosa. Methods. PCR amplification, cloning and sequencing of 5S rDNA, bioinformatics analysis. Results. The 5SrDNA IGS of P. spinosa was cloned sequenced and compared with the IGS of three other representatives of the genus Prunus. It was found that the IGS variants present in the genome of P. spinosa differ by the number of subrepeats located at the 5’end. Each of these subrepeats contain RNA Pol III transcription termination signal. The IGS sequences of Prunus species evolved mainly by accumulation of nucleotide substitutions. The level of intragenomic similarity of P. spinosa IGS is 96.5-100%, while the similarity between the IGS of different Prunus species ranges from 73.0 to 87.3%. Conclusions. The IGS of 5S rDNA represents a useful marker for phylogenetic and taxonomic studies in the genus Prunus.Keywords: 5S rDNA, molecular evolution, Prunus spinosa.

Molecular organization of 5S ribosomal DNA of Apis mellifera ligustica

Aim. 5S rDNA belongs to the moderately repeated, tandemly arranged sequences composed of coding regions and intergenic spacers (IGS). The IGS comparison is successfully used for the study of microevolution and in the molecular taxonomy of plants, vertebrates and some invertebrates. However, 5S rDNA of insects still remains insufficiently characterized. In this article, we analyze the molecular organization and polymorphism of 5S rDNA IGS of Apis mellifera ligustica. Methods. DNA extraction, PCR amplification, cloning of 5S rDNA, sequencing, bioinformatics analysis. Results. The 5S rDNA IGS of A. m. ligustica was cloned into the plasmid vector and sequenced. The obtained sequences were compared with the same genomic region of A. m. carnica. It was found that the genome of A. m. ligustica contains one class of IGS, which is represented by structural subclasses 1A, 1B, 1G and 1F. Two of these subclasses, 1G and 1F, which are identified here for the first time, are specific for A. m. ligustica. Conclusions. The obtained results show a high intra- and intergenomic polymorphism of 5S rDNA within the subspecies of Apis mellifera.Keywords: 5S rDNA, intergenic spacer, repeated sequences, Apis mellifera, Apidae.

Ecology of microorganisms from springs of national park "Alkhanai" (Transbaikalia, Russia)

The composition and diversity of microbial communities from the cold ultra-fresh oligotrophic seeps and River Sukhoe Ubzhogoe of the National Park "Alkhanai" were determined via culture-dependent vs. culture-independent approaches. The microbial community of six groundwater seeps determined by 16S rDNA cloning, and sequencing consisted of four dominant genera belonging to the classes Betaproteobacteria and Gammaproteobacteria; they were Duganella sp., Janthinobacterium sp., and Pseudomonas sp., Serratia sp., respectively. Uncultured bacteria were presented by the phylum Bacteroidetes, classes Flavobacteriia (Flavobacterium sp.), Cytophagia (Flexibacter sp.), and Chitinophagia (Sediminibacterium sp.); the phylum Proteobacteria, classes Betaproteobacteria (Comamonadaceae) and Gammaproteobacteria (Acinetobacter sp.); and the phylum Actinobacteria, class Actinomycetia (Rhodococcus sp.). The same chemoorganoheterotrophic bacteria were cultivated as dominant species from the waters of the investigated seeps. Still, differences in the microbial communities were found in a proper combination of the four dominant forms of bacteria in the appropriate ecosystems. One of the peculiarities of the cultivable bacteria was the production of colored pigments violacein and prodigiosin, known for their therapeutic properties. The subject is promising for fundamental and applied research. Curative properties of cold seeps were discussed, and it was proposed that the water quality could assume medicinal qualities due to the specific composition of microorganisms.

Investigation of microbial community changes in petroleum polluted sediments during hydrocarbons degradation

ABSTRACT Bioremediation is among the most basic and reliable way to eliminate pollutants by transforming the toxic petroleum components into less toxic metabolites. In order to compare the change of bacterial community during oil-biodegradation process, two oil-polluted sediments sampled from Tunisia were analyzed using 16S rDNA clone libraries. During the experimental period, measurements of microbial abundance along with qualitative and quantitative analysis of pollutants were carried out. Fingerprinting analysis of natural samples showed the presence of different classes of bacteria with the dominance of Gammaproteobacteria (>60%). The bacterial diversity decreased in enrichment cultures with crude oil with the appearance and predominance of Alcanivorax spp. Our data also showed that several Obligate Marine Hydrocarbonoclastic Bacteria (OMHB: Alcanivorax, Cycloclasticus and Marinobacter) were stimulated by the addition of petroleum. The shift of diversity and the dominance of OMHB were associated with high degradation rate of crude oil (the total degradation of petroleum TERHCs was estimated at 90% and 70% in TST1E and TST2E) and a decrease in sediment toxicity to Corophium orientale. Since several factors can influence the clean-up in-field, the effectiveness of biostimulation treatment and its optimization at lab scale under controlled conditions would facilitate and allow the application in-situ. Moreover, the study of the community diversity allows the detection and the identification of the dominant hydrocarbonoclastic species such as Alcanivorax which it would be beneficial in future experiments to facilitate the selection of the autochthonous strains (adapted to support the contamination) for the reconstruction of efficient consortia useful in the cleanup of hydrocarbon contaminated sites.

Microbiota of a UASB Reactor Treating Palm Oil Mill Effluent Using HiSeq Sequencing

Potential microbial communities in the UASB reactor fed with two different substrates i.e synthetic wastewater and raw palm oil mill effluent (POME) were elucidated by using one of the most popular techniques in molecular methods, viz 16S rDNA cloning. The methodology involved; the extraction of nucleic acids, amplification and cloning of the 16S rRNA genes on sequencing HiSeq platform and finally identification and affiliation of the isolated clone with the aid of phylogenetic software. Results showed that the genus methanosarcina and methanosaeta were dominant methanogens in this study for both substrates types. Overall, microbial population (Bacteria and Archaea) in sample A (POME as substrate) is more diverse compared to sample B (synthetic wastewater as substrate) due to abundance of microorganism population in raw POME which was used as a substrate. However for the methanogenic (Archaea) diversity in both samples, there was not much different between sample A and sample B probably due to similar inoculum was inoculated in the reactor despite of have using different substrate type.

Contrasting endolithic habitats for cyanobacteria in spring calcites of the European Alps.

It is often difficult to decide which cyanobacteria found in endolithic habitats of calcite spring-tufa deposits are present as ephemeral components of the biota or are persistent, structural elements. To answer this question, we repeatedly studied two microhabitats of contrasting calcareous tufa springs in the European Alps. Pigment extracts, fluorescence probe measurements of in situ samples and traditional microscopy confirmed the dominance of cyanobacteria over eukaryotic algae and their viability in both microhabitats. Spring Site 1 (Laas, Northern Italy) is characterized by a highly variable, moist to dry and sun-exposed waterfall tufa consisting of fibrous calcite. A segment of these deposits in the lateral flank of a grotto contained dark endolithic layers in dim light, 1-2 mm below the surface, where aggregated cyanobacterial cells were dominant but not directly attached to calcites, a potential sign of gentle endolithic dissolution rather than calcite precipitation induced by cyanobacteria. Site 2 (Mühlau, Austria), in contrast, is a moss-tufa microhabitat associated with a seepage spring situated in a shady gorge, where the targeted stromatolites consisted of bark-like sheets of friable, orange to light-brown when wet (drying violet) 'styrofoam'- like aggregates of minute crystallites on the day-light exposed surfaces. These calcites were observed to nucleate directly on external sheaths of viable cyanobacteria trichomes. A polyphasic approach including LM, SEM, TEM exhibited a number of identical but also some divergent cyanobacteria of which two key taxa were specific for each of the two microhabitats (Nostoc and Pseudoscytonema at Sites 1 and 2 respectively). Both cyanobacterial communities characterised, by the cloning of 16S rDNA showed a dominance of mostly unknown and partly divergent filamentous cyanobacteria assigned to the order of Synechococcales. Our microhabitat study of alpine crenal calcites highlights the rather divergent biotic responses of cyanobacteria within spring tufa deposits.

Organization of 5S ribosomal DNA of Litchi chinensis Sonn.

Aim. 5S ribosomal DNA (5S rDNA) represents a universal model for studying the evolution of repeated sequences in eukaryotic organisms. Taking into account that this region of the genome still remains almost undescribed in species of the family Sapindaceae, we investigated the molecular organization of a repeated unit of 5S rDNA in a member of this family, Litchi chinensis. Methods. PCR amplification, cloning and sequencing of 5S rDNA. Results. It was found that the length of the repeated unit of the 5S rDNA of L. chinensis is 321 -323 bp. The level of intragenomic similarity of 5S rDNA repeats is 87.1 %. Potential external elements of the RNA polymerase III promoter, which are localized in IGS, differ from those described for members of other families of angiosperms. Conclusions. In the genome of L. chinensis, at least two classes of 5S rDNA repeats are present, which differ in the sequence of external promoter elements.
 Keywords: 5S rDNA, molecular evolution, Litchi chinensis, Sapindaceae.

Study on the community structure and function of anaerobic granular sludge under trichloroethylene stress.

Trichloroethylene (TCE) is one of the most common groundwater pollutants. It is carcinogenic, teratogenic, mutagenic and poses a serious threat to human health and the environment. Therefore, reducing the environmental toxicity of TCE is of great significance. Anaerobic sludge was cultured and acclimated in an upflow anaerobic sludge blanket (UASB) reactor in this study. The Chemical Oxygen Demand (COD) concentration of the influent was approximately 2500 mg L-1, and the TCE concentration of the influent ranged from 1.46 mg L-1 to 73 mg L-1. After biodegradation of the anaerobic microflora, the COD removal rate was approximately 85%, and the TCE removal rate was over 85%. The microbial community of anaerobic sludge was analysed by 16 S rDNA clone libray and 454 high-throughput sequencing. Through analysis of the sequencing results, we found that there were a variety of acid-forming bacteria, anaerobic dechlorinating bacteria, and methanogenic bacteria. Based on the analysis of microflora function, it was speculated that the TCE metabolic pathway took place in UASB reactors. Desulfovibrio and Syntrophobacter provided an anaerobic environment, and acid-forming bacteria metabolise organic compounds into hydrogen. With Dehalobacter and Geobacter, TCE as an electron acceptor is dechlorinated and reduced under the anaerobic environment, in which hydrogen acts as an electron donor. By this, we clarified the metabolic pathway for improving TCE bioremediation.

Molecular Characterization of Bacterial Community Composition in the Rhizosphere of Invasive Plant Species Amur Honeysuckle (<i>Lonicera maackii</i>) in an Urban Wetland Forest

The goal of this research was to determine the effects of the growth of invasive plant Amur Honeysuckle (Lonicera maackii) on the rhizosphere bacterial community composition, and diversity in an urban wetland forest ecosystem. Bacterial communities from the rhizosphere of 5 L. maackii plants and control bulk soils that did not have any L. maackii were investigated at Nina Mason Pulliam EcoLab (NMPE) using a culture-independent pipeline. Bacterial communities were characterized by PCR amplification and cloning 16S rRNA gene fragments following total DNA isolation from the soil samples. Microbial communities associated with both L. maackii rhizosphere and control sites showed high bacterial diversity within each site and taxa unique to individual sites were observed. Phylogenetic analyses revealed 80% of 400 16S rDNA clones were classified as α-, β- and γ-Proteobacteria, Acidobacteria, Actinobacteria, Cytophaga-Flexibacter-Bacteroides (CFB) group, and Verrucomicrobia. Members of the Proteobacteria and Acidobacterium represented 66.5% and 14.5% of the clone library, respectively, whereas the remaining bacterial divisions each comprised less than 7% of the clone library. Twenty-five 16S rDNA clones could not be classified into any known bacterial divisions. Statistical analyses showed significant differences in the presence of L. maackii on the proportions of 16S rDNA clones affiliated with Proteobacteria and Acidobacterium, suggesting bacterial community composition and structure does significantly change in the presence of L. maackii. However, sequence-based community analysis and the corresponding lack of intact microbial cultures limit understanding of the potential influences of enriched microbial taxa on plant hosts and their roles in ecosystem functioning.

Molecular Organization of 5S Ribosomal DNA and Taxonomic Status of Avenella flexuosa (L.) Drejer (Poaceae)

The wavy hair-grass, Avenella flexuosa (L.) Drejer (also known as Deschampsia flexuosa (L.) Trin. or Lerchenfeldia flexuosa (L.) Schur), is a species of perennial herbaceous plant of the Poaceae family that is widespread in Eurasia, Africa, North and South America. Whether this species belongs to the Deschampsia genus has long been an open question. Taking into account that the 5S rDNA comparison is a convenient tool in the molecular taxonomy of plants, we cloned and sequenced this region of the A. flexuosa genome and evaluated the genetic distances between this species and other species of the Poeae tribe. It has been found that at least two structural classes of 5S rDNA repeats are present in the A. flexuosa genome. The spacer regions in the 5S rDNA of A. flexuosa and D. antarctica have a low level of similarity that does not exceed the level of similarity with representatives of several other genera of the Poeae tribe. The 5S rDNA clones of A. flexuosa and D. antarctica form two distinct clades on the phyllodendogram. Taking together, our results support the idea that A. flexuosa belongs to a separate genus Avenella.

Isolation and identification of contaminant microorganisms in industrial and non-industrial Iranian fermented milk (Doogh)

Doogh is a traditional fermented milk based drink, that is a very popular product in Iran, its microbial safety is important. The purpose in this study, 16S based rDNA PCR, cloning and sequencing was to investigated the exploration microbial contaminants in commercial pasteurized Doogh specimens. To isolate and identify the contaminants in 24 different Doogh samples (17 different industrial and 6 different non-industrial samples) Polymerase chain reaction (PCR) was applied. The experimental results revealed that lactic acid bacteria (Lactobacillus brevis, L. fermentum, L. paracasei and L. gallinarum) gram-positive spore-forming bacteria (Bacillus anthracis, B. licheniformis. and B. subtilis) and acetic acid bacteria (Acetobacter indonesiensis and A. tropicalis) were identified in Doogh samples. Likewise, result obtained from sequencing of D1/D2 26S rDNA determined the presence of different yeasts including Saccaromyces sp. (S. unisporous, S. pastorianus) Kazachstaia sp. (K. servazzi, K. unispora) Pichia fermentans, Galctomyces candidum, and Cryptococcus magnus. The results demonstrated that some types of bacteria and yeasts and molds were identified as the main contaminants in Iranian Doogh.