Comparison of the Bacterial Microbiota in a Bale of Collected Cardboard Determined by 454 Pyrosequencing and Clone Library

Biofouling, the accumulation of microorganisms, is a major problem in paper mills processing paper and cardboard. This leads to the production of lower quality recycled products. Several studies have focused on the microbial content in the paper mill and the final products. Our aim was to determine the microbial biota in a bale of collected cardboard prior to entering the paper mill. Total genomic DNA was isolated and analyzed using two different methods for comparison purposes: 454 pyrosequencing and clone library. A total of 3268 V6-V8 454 pyrosequencing reads and 322 cloned V6-V8 16S rRNA nucleotide sequences were obtained. Both methods showed the presence of three major bacterial genera: Bacillus, Solibacillus and Paenibacillus, all members of the spore-forming phylum Firmicutes. Pyrosequencing, however, revealed a richer and more diverse bacterial community than clone library. It showed the presence of additional minor Firmicute genera and of a small number of Proteobacteria. The sorting at the recycling plant, the storing, and the processing at the paper mill, the end uses, will all contribute to the bacterial microbiota present in a bale of collected cardboard as revealed here.

CONSTRUCTION AND SIGNIFICANCE OF DIRECTIONAL cDNA EXPRESSION LIBRARY FROM HUMAN MYELOMA CELL

Objective To construct human myeloma cell cDNA expression library as to screen myeloma tumor antigen. Methods Total RNA and purified mRNA were extracted from human myeloma cell line HMy2. First and second strand cDNA were synthesized through reverse transcription. After blunting, the cDNA fragments were ligated with EcoR I adapters. Then the cDNAs were digested by Xho I, and smaller than 400bp were removed by Sephacryl-S400 spin column, the remaining were ligated with λZAP vector. The recombinants were packaged in vitro, and a small portion of packaged phage was used to infect E.coli XL1-Blue-MRF for titration. The recombinants were examined by color selection. In order to evaluate the size of cDNA inserts and the diversity of library, the pBK-CMV phagemid were excised from the ZAP express vector by using ExAssist helper phage with XLOLR strain , and then the pBK-CMV phagemid were digested by Xho I and EcoR I. Results The HMy2 cell line cDNA library consisting of 1.58×10 6 recombinant bacteriophages was constructed with the recombinant ratio 99.6%. The average length of the recombinant exogenous inserts was about 1.7kb.Conclusion The constructed cDNA library are deserved to screen target clones.

Archaeal and bacterial communities in acid mine drainage from metal-rich abandoned tailing ponds, Tongling, China

To expand knowledge on microbial communities of various metal-rich levels of mine drainage environments in Anhui province, China, the archaeal and bacterial diversities were examined using a PCR-based cloning approach. Eight acid mine water samples were collected from five areas in Tongling. Phylogenetic analyses revealed that bacteria mainly fell into ten divisions, which were Betaproteobacteria, Gammaproteobacteria, Alphaproteobacteria, Deinococcus-Thermus, Nitrospira, Firmicutes, Actinobacteria, Deltaproteobacteria, Bacteroidetes, Chloroflexi. Archaea fell into three phylogenetic divisions, Thermoplasma, Ferroplasma and Thermogymnomonas. The unweighted pair group method with arithmetic mean（UPGMA） cluster analysis based on the microbial communities’ compositions revealed that five samples shared similarity with the dominance of Meiothermus and Thermomonas. Two samples had the preponderant existence of Acidithiobacillus and Leptospirillum. The remaining sample owned higher microbial communities’ diversity with the Shannon-Weaver H up to 2.91. Canonical correlation analysis（CCA） suggested that microbial community structures had great association with p H and the concentration of Hg2＋, Pb2＋, Fe3＋, Cl-, SO2- 4in water.

Assessing the impact of fungicide enostroburin application on bacterial community in wheat phyllosphere

Fungicides have been used extensively for controlling fungal pathogens of plants. However, little is known regarding the effects that fungicides upon the indigenous bacterial communities within the plant phyllosphere. The aims of this study were to assess the impact of fungicide enostroburin upon bacterial communities in wheat phyllosphere. Culture-independent methodologies of 16S rDNA clone library and 16S rDNA directed polymerase chain reaction with denaturing gradient gel electrophoresis （PCR-DGGE） were used for monitoring the change of bacterial community. The 16S rDNA clone library and PCR-DGGE analysis both confirmed the microbial community of wheat plant phyllosphere were predominantly of the γ-Proteobacteria phyla. Results from PCR-DGGE analysis indicated a significant change in bacterial community structure within the phyllosphere following fungicide enostroburin application. Bands sequenced within control cultures were predominantly of Pseudomonas genus, but those bands sequenced in the treated samples were predominantly strains of Pantoea genus and Pseudomonas genus. Of interest was the appearance of two DGGE bands following fungicide treatment, one of which had sequence similarities （98%） to Pantoea sp. which might be a competitor of plant pathogens. This study revealed the wheat phyllosphere bacterial community composition and a shift in the bacterial community following fungicide enostroburin application.

Microbial Community Dynamics During Biogas Slurry and Cow Manure Compost

This study evaluated the microbial community dynamics and maturation time of two compost systems： biogas slurry compost and cow manure compost, with the aim of evaluating the potential utility of a biogas slurry compost system. Denaturing gradient gel electrophoresis （DGGE）, gene clone library, temperature, C/N ratio, and the germination index were employed for the investigation, cow manure compost was used as the control. Results showed that the basic strip and dominant strips of the DGGE bands for biogas slurry compost were similar to those of cow manure compost, but the brightness of the respective strips for each system were different. Shannon-Weaver indices of the two compost systems differed, possessing only 22% similarity in the primary and maturity stages of the compost process. Using bacterial 16S rRNA gene clone library analysis, 88 bacterial clones were detected. Further, 18 and 13 operational taxonomic units （OTUs） were present in biogas slurry and cow manure compost, respectively. The 18 OTUs of the biogas slurry compost belonged to nine bacterial genera, of which the dominant strains were Bacillus sp. and Carnobacterium sp.; the 13 OTUs of the cow manure compost belonged to eight bacterial genera, of which the dominant strains were Psychrobacter sp., Pseudomonas sp., and Clostridium sp. Results demonstrated that the duration of the thermophilic phase （more than 50°C） for biogas slurry compost was 8 d less than the according duration for cow manure compost, and the maturation times for biogas slurry and cow manure compost were 45 and 60 d, respectively. It is an effective biogas slurry assimilate technology by application of biogas slurry as nitrogen additives in the manufacture of organic fertilizer.

Soil fungistasis and its relations to soil microbial composition and diversity:A case study of a series of soils with different fungistasis

Fungistasis is one of the important approaches to control soil-borne plant pathogens.Some hypotheses about the mechanisms for soil fungistasis had been established,which mainly focused on the soil bacterial community composition,structure,diversity as well as function.In this study,the bacterial community composition and diversity of a series of soils treated by autoclaving,which coming from the same original soil sample and showing gradient fungistasis to the target soil-borne pathogen fungi Fusarium grami...

Molecular analysis of microbial community in a groundwater sample polluted by landfill leachate and seawater

Seashore landfill aquifers are environments of special physicochemical conditions (high organic load and high sa- linity), and microbes in leachate-polluted aquifers play a significant role for intrinsic bioremediation. In order to characterize microbial diversity and look for clues on the relationship between microbial community structure and hydrochemistry, a cul- ture-independent examination of a typical groundwater sample obtained from a seashore landfill was conducted by sequence analysis of 16S rDNA clone library. Two sets of universal 16S rDNA primers were used to amplify DNA extracted from the groundwater so that problems arising from primer efficiency and specificity could be reduced. Of 74 clones randomly selected from the libraries, 30 contained unique sequences whose analysis showed that the majority of them belonged to bacteria (95.9%), with Proteobacteria (63.5%) being the dominant division. One archaeal sequence and one eukaryotic sequence were found as well. Bacterial sequences belonging to the following phylogenic groups were identified: Bacteroidetes (20.3%), β, γ, δ and ε-subdivisions of Proteobacteria (47.3%, 9.5%, 5.4% and 1.3%, respectively), Firmicutes (1.4%), Actinobacteria (2.7%), Cyanobacteria (2.7%). The percentages of Proteobacteria and Bacteroides in seawater were greater than those in the groundwater from a non-seashore landfill, indicating a possible influence of seawater. Quite a few sequences had close relatives in marine or hypersaline environments. Many sequences showed affiliations with microbes involved in anaerobic fermentation. The remarkable abundance of sequences related to (per)chlorate-reducing bacteria (ClRB) in the groundwater was significant and worthy of further study.

Bacterial diversity in activated sludge from a consecutively aerated submerged membrane bioreactor treating domestic wastewater

The bacterial diversity of activated sludge from submerged membrane bioreactor (SMBR) was investigated. A 16S rDNA clone library was generated, and 150 clones were screened using restriction fragment length polymorphism (RFLP). Of the screened clones, almost full-length 16S rDNA sequences of 64 clones were sequenced. Phylogenetic tree was constructed with a database containing clone sequences from this study and bacterial rDNA sequences from NCBI for identification purposes. The 90.6% of the clones were a?l...

Comparison of Bacterioplankton Communities in Three Heavily Polluted Streams in China

Objective To compare the bacterioplankton communities in streams exposed to pollution of different types. Methods The bacterioplankton communities in three selected heavily polluted streams were investigated by using terminal‐restriction fragment length polymorphism （T‐RFLP） analysis in combination with 16S rRNA gene clone library analysis. Results Both T‐RFLP and 16S rRNA gene clone library revealed a great difference in bacterioplankton community composition in the different streams. Conclusion This work might provide some new insights into bioremediation of heavily polluted streams.

Phylogenetic diversity of ribulose-1, 5-bisphosphate carboxylase/oxygenase large subunit genes of bacterioplankton in the East China Sea

Phylogenetic diversity of Form I and Form II ribulose1, 5-bisphosphate carboxylase/oxygenase (RubisCO) largesubunit (rbcL) genes in the inshore and offshore areas of the East China Sea were investigated. Two new primer setswere designed for amplifying partial sequences of rbcL genes from Proteobacteria. Four rbcL gene clone libraries wereconstructed by amplification and cloning of approximately 640~800 bp sequences of bacterioplankton populations.The method of screening library by denaturing gradient gel electrophoresis (DGGE) was introduced. The resultsshow that the diversity of Form I is higher in offshore waters with higher salinity and lower productivity, while thatof Form II is higher at the inshore station where salinity is lower and productivity is higher. Several clusters ofsequences obtained are deeply rooted and show low similarity (60%~78%) to the known rbcL in existing databases.The degree of diversity of rbcL genes is directly related to environmental variables, including temperature, salinity,pH, dissolved oxygen, etc. These results indicate that rbcL gene can be used as an effective indicator for geneticdiversity and population variability of bacterioplankton with the ability of carbon dioxide fixation in the sea.

Biodegradation ofEnteromorpha Polysaccharides by Intestinal Micro-Community from Siganus oramin

Micro-communities are supposed to have more potential functions of biodegradation of polysaccharides than single strain; however, the intestinal mi ties involved in the biodegradation of Enteromorpha polysaccharides （EP） were sel- dom reported. In order to obtain the EP-degrading micro-community, the intestines of Siganus oramin was obtained to isolate the micro-communities, which were enriched by 0.3% of EP as the sole carbon source. A stable micro-community with EP degradative capability was achieved after seven generations of subculture, named H1. Results showed that H1 was able to degrade 75% of EP within 24 hours, and the activity of EP lyases reached 500 U mL-1 in 32 hours. With denaturing gradient gel electrophoresis （DGGE） and 16S rRNA gene clone library analysis, ten bacteria closely related to Marinomonas pontica, Microbacterium sp., Leucobacter chironomi, Cyclobacterium sp., Algoriphagus winogradskyi, Pseudoalteromonas sp. and Vibrio sp. were determined. Furthermore, compared with the DGGE bands sequence and the clone library analysis, the dominant bacteria of the EP-biodegrading mi- cro-community were Pseudoalteromonas sp. and Vibrio sp., with the respective proportion of 38% and 46%, and they should play an important role in EP degradation together with other degrading bacteria in the micro-community H1.

Phylogenetic diversity of planktonic bacteria in the Chukchi Borderland region in summer

Planktonic bacteria are abundant in the Chukchi Borderland region. However, little is known about their di- versity and the roles of various bacteria in the ocean. Seawater samples were collected from two stations K2S and K4S where sea ice was melting obviously. The analysis of water samples with fluorescence in situ hybridization （FISH） showed that DMSP-degrading bacteria accounted for 13% of the total bacteria at the station K2S. No aerobic anoxygenic phototrophic （AAP） bacteria were detected in both samples. The bacterial communities were characterized by two 16S rRNA gene clone libraries. Sequences fell into four major lineages of the domain Bacteria, including Proteobacteria （Alpha, Beta and Gamma subclasses）, Bac- teroidetes, Actinobacteria and Firmicutes. No significant difference was found between the two clone li- braries. SAR11 and Rhodobacteraceae clades of Alphaproteobacteria and Pseudoalteromonas of Gammapro- teobacteria constituted three dominant fractions in the clone libraries. A total of 191 heterotrophic bacterial strains were isolated and 76% showed extracellular proteolytic activity. Phylogenetic analysis reveals that the isolates fell into Gammaproteobacteria, Bacteroidetes, Actinobacteria and Firmicutes. The most common genus in both the bacterial isolates and protease-producing bacteria was Pseudoalteromonas. UniFrac data showed suggestive differences in bacterial communities between the Chukchi Borderland and the northern Bering Sea.

Development and evaluation of specific PCR primers targeting the ribosomal DNA-internal transcribed spacer(ITS)region of peritrich ciliates in environmental samples

Peritrich ciliates are highly diverse and can be important bacterial grazers in aquatic ecosystems. Morphological identifi cations of peritrich species and assemblages in the environment are time-consuming and expertise-demanding. In this study, two peritrich-specifi c PCR primers were newly designed to amplify a fragment including the internal transcribed spacer(ITS) region of ribosomal rDNA from environmental samples. The primers showed high specifi city in silico, and in tests with peritrich isolates and environmental DNA. Application of these primers in clone library construction and sequencing yielded exclusively sequences of peritrichs for water and sediment samples. We also found the ITS1, ITS2, ITS, D1 region of 28 S rDNA, and ITS+D1 region co-varied with, and generally more variable than, the V9 region of 18 S rDNA in peritrichs. The newly designed specifi c primers thus provide additional tools to study the molecular diversity, community composition, and phylogeography of these ecologically important protists in dif ferent systems.

Dynamic genetic features of eukaryotic plankton diversity in the Nakdong River estuary of Korea

Estuaries are environments where freshwater and seawater mix and they display various salinity profiles.The construction of river barrages and dams has rapidly changed these environments and has had a wide range of impacts on plankton communities.To understand the dynamics of such communities,researchers need accurate and rapid techniques for detecting plankton species.We evaluated the diversity of eukaryotic plankton over a salinity gradient by applying a metagenomics tool at the Nakdong River estuary in Korea.Environmental samples were collected on three dates during summer and autumn of 2011 at the Eulsukdo Bridge at the mouth of that river.Amplifying the 18 S rDNA allowed us to analyze 456 clones and 122 phylotypes.Metagenomic sequences revealed various taxonomic groups and cryptic genetic variations at the intra-and inter-specific levels.By analyzing the same station at each sampling date,we observed that the phylotypes presented a salinity-related pattern of diversity in assemblages.The variety of species within freshwater samples reflected the rapid environmental changes caused by freshwater inputs.Dinophyceae phylotypes accounted for the highest proportion of overall diversity in the seawater samples.Euryhaline diatoms and dinoflagellates were observed in the freshwater,brackish and seawater samples.The biological data for species composition demonstrate the transitional state between freshwater and seawater.Therefore,this metagenomics information can serve as a biological indicator for tracking changes in aquatic environments.

Eubacteria and Archaea community of simultaneous methanogenesis and denitrification granular sludge

Based on the successful performance of a lab-scale upflow anaerobic sludge blanket （UASB） reactor with the capacity of simultaneous methanogenesis and denitrification （SMD）, the specific phylogenetic groups and community structure of microbes in the SMD granule in the UASB reactor were investigated by the construction of the Eubacteria and Archaea 16S rDNA clone libraries, fragment length polymorphism, and sequence blast. Real time quantitative-polymerase chain reaction （RTQ-PCR） technique was used to quantify the contents of Eubacteria and Archaea in the SMD granule. The contents of some special predominant methanogens were also investigated. The results indicated that the Methanosaeta and Methanobacteria were the predominant methanogens in all Archaea in the SMD granule, with contents of 71.59% and 22.73% in all 88 random Archaea clones, respectively. The diversity of Eubacteria was much more complex than that of Archaea. The low GC positive gram bacteria and ε-Protebacteria were the main predominant Eubacteria species in SMD granule, their contents were 49.62% and 12.03% in all 133 random Eubacteria clones respectively. The results of RTQ-PCR indicated that the content of Archaea was less than Eubacteria, the Archaea content in total microorganisms in SMD granule was about 27.6%.

Microbial community structure and diversity in deep-sea hydrothermal vent sediments along the Eastern Lau Spreading Centre

The aim of this study is to investigate microbial structures and diversities in five active hydrothermal fields＇ sediments along the Eastern Lau Spreading Centre （ELSC） in the Lau Basin （southwest Pacific）. Microbial communities were surveyed by denatured gradient gel electrophoresis （DGGE） and clone library analysis of 16S rRNA genes. The differences in microbial community structures among sediment samples from the five deep-sea hydrothermal sites were revealed by DGGE profiles. Cluster analysis of DGGE profiles sepa- rated the five hydrothermal samples into two groups. Four different 16S rRNA gene clone libraries, repre- senting two selected hydrothermal samples （19-4TVG8 and 19-4TVG11）, were constructed. Twenty-three and 32 phylotypes were identified from 166 and 160 bacterial clones respectively, including Proteobacteria, Bacteroidetes, Firmicutes, Nitrospirae and Planctomycetes. The phylum Proteobacteria is dominant in both bacterial libraries with a predominance of Gamma-Proteobacteria. A total of 31 and 25 phylotypes were obtained from 160 and 130 archaeal clones respectively, including Miscellaneous Crenarchaeotic Group, Marine Group Ⅰ and Ⅲ, Marine Benthic Group E, Terrestrial Hot Spring Crenarchaeota and Deep-sea Hy- drothermal Vent Euryarchaeota. These results show a variety of clones related to those involved in sulfur cycling, suggesting that the cycling and utilization of sulfur compounds may extensively occur in the Lau Basin deep-sea hydrothermal ecosystem.

Characterization of the airborne bacteria community at different distances from the rotating brushes in a wastewater treatment plant by 16S rRNA gene clone libraries

Biological risks of bioaerosols emitted from wastewater treatment processes have attracted wide attention in the recent years. However, the culture-based analysis method has been mostly adopted for detecting the bacterial community in bioaerosols, which may result in the underestimation of total microorganism concentration as not all microorganisms are cultivable. In this study, oligonucleotide fingerprinting of 16S rRNA genes was applied to reveal the composition and structure of the bacterial community in bioaerosols from an Orbal oxidation ditch in a Beijing wastewater treatment plant （WWTP）. Bioaerosols were collected at different distances from the aerosol source, rotating brushes, and the sampling height was 1.5 m which is the common respiratory height of a human being. The bacterial communities of bioaerosols were diverse, and the lowest bacterial diversity was found at the sampling site just after the rotating brush rotating brush. A large proportion of bacteria in bioaerosols were affiliated with Proteobacteria and Bacteroidetes. Numerous bacteria present in the bioaerosols also emerged in water, indicating that the bacterial community in the bioaerosols was related to that of the aerosols＇ sources. The forced aeration of rotating brushes brought about observably distinct bacterial communities between sampling sites situated before and after the rotating brush. Isolation sources of closest relatives in bioaerosols clone libraries were associated with the aqueous environment in the WWTP. Common potential pathogens in bioaerosols as well as those not reported in previous research were also analyzed in this study. Measures should be adopted to reduce the emission of bioaerosols and prevent their exposure to workers.

Construction and primary characterization of Echinococcus multilocularis protoscolex cDNA expression library

Objective To construct a λgt11 cDNA expression library of Echinococcus multilocularis protoscolex isolated in China. Methods Echinococcus multilocularis protoscolex mRNA was extracted using a Quickprep MicromRNA purification kit based on combining of the disruptive and protective properties of guanidinium thiocyanate (GTC) with the speed and selectivity of oligo (dT)-cellulose chromatography in a spum-column with some modification. Purified mRNA (1.8 μg) was submitted to reverse transcription using random hexamers ［pd(N6)］. The double-strand blunt-ended cDNAs were ligated with an EcoRI/NotI adaptor to form a cohesive EcoRI end. Subsequently the synthesized cDNA was inserted into vector λgt11 EcoRI arms. After being packaged in vitro, λgt11 was put to an infectious bacteria Echinococcus coli (E.coli) strain Y1090; the recombinants were screened by color selection. PCR amplification was performed to evaluate the size of insertion DNA fragments.Results The recombinant ratio was nearly 100% and approximately 1×106 clones could be derived from this λgt11 cDNA library. PCR results indicated that the insertion DNAs were about 1.48 kb. Conclusions A λgt11 cDNA expression library consisting of a million recombinant clones has been constructed from Echinococcus multicularis protoscolex mRNA. Further studies on this library are deserved.

Analysis of the Fungal Community in Apple Replanted Soil Around Bohai Gulf

Apple replant disease(ARD) is a frequently occurring plant disease in replanted orchards around Bohai Gulf, which causes growth inhibition and even death of plants. The aim of this study was to investigate the etiology of ARD around Bohai Gulf. In this study, the primary growth inhibition of apple seedlings was evaluated in ten replanted soils, sampled around Bohai Gulf. A fungal clone library was used to identify changes in the structure and composition of the soil fungal community. The results revealed that the Simpson diversity indices of Laizhou and Pulandian orchards were higher than others, presenting severe ARD. Ascomycota dominated around Bohai Gulf at the phyla level. Fusarium and Saccharomyces were abundant in all replanted soils. In addition, correlations between the relative abundance of fungal genera in soils and the severity of ARD were analyzed. The results showed that Fusarium was correlated positively with the severity of ARD, but Mortierella was negatively correlated. Furthermore, the quantitative PCR of Fusarium oxysporum, which was regarded as a factor of ARD, was performed. Overall, this study demonstrated that ARD was strongly associated with an unbalanced microbial ecosystem with more pathogenic fungi, while Fusarium in the apple replanted soil was the key factor for ARD around Bohai Gulf.

Analysis of bacterial community in bulking sludge using culture-dependent and -independent approaches

The bacterial community of a bulking sludge from a municipal wastewater treatment plant with anoxic-anaerobic-oxic process was investigated by combination of cultivation and 16S rRNA gene clone library analysis for understanding the causes of bulking.A total of 28 species were obtained from 63 isolates collected from six culture media.The most cultivable species belonged to γ-Proteobacteria including Klebsiella sp.,Pseudomonas sp.,Aeromonas sp.and Acinetobacter sp.Further analysis of these strains by repetitive sequence based on polymerase chain reaction （rep-PCR） technology showed that rep-PCR yielded discriminatory banding patterns within the same genus using REP and BOX primer sets.While the culture-independent assessment revealed that β-Proteobacteria was the dominant group in the bulking sample.Sequence analysis revealed that the highest proportion （14.7%） of operational taxonomic units was 98% similar to Candidatus Accumulibacter phosphatis,which is used to remove phosphorous from wastewater.Our results indicated that combining different approaches can produce complementary information,thus generate a more accurate view of microbial community in bulking sludge.