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.

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.

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.

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...

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.

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.

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.

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.

Archaeal community structure along a gradient of petroleum contamination in saline-alkali soil

The response of archaeal communities to petroleum contamination in saline-alkali soil was characterized by analyses of three soil samples with different total petroleum hydrocarbon concentrations.Through the construction and screening of 16S rRNA gene clone libraries based on DNA extracts from these soils,nine distinct phylogenetic groups were identified.Statistical analyses showed that the distribution of archaeal community structures differ significantly along the gradient of petroleum contamination in these three saline- alkali soils.Five phylogenetic groups were dominant in the control soil,two of which were also abundant in the lightly contaminated soil.Four phylogenetic groups were dominant in heavily contaminated soil,one of which was also abundant in the lightly contaminated soil.The halophilic genus of Haloferax and the haloalkaliphilic genus of Natronomonas were more abundant in heavily contaminated soil.These results suggested that the genera of Haloferax and Natronomonas may have a role in the natural attenuation of petroleum- contaminated saline-alkali soil.

Microbial community structures in an integrated two-phase anaerobic bioreactor fed by fruit vegetable wastes and wheat straw

The microbial community structures in an integrated two-phase anaerobic reactor（ITPAR）were investigated by 16 S r DNA clone library technology. The 75 L reactor was designed with a 25 L rotating acidogenic unit at the top and a 50 L conventional upflow methanogenic unit at the bottom, with a recirculation connected to the two units. The reactor had been operated for 21 stages to co-digest fruit/vegetable wastes and wheat straw, which showed a very good biogas production and decomposition of cellulosic materials. The results showed that many kinds of cellulose and glycan decomposition bacteria related with Bacteroidales,Clostridiales and Syntrophobacterales were dominated in the reactor, with more bacteria community diversities in the acidogenic unit. The methanogens were mostly related with Methanosaeta, Methanosarcina, Methanoculleus, Methanospirillum and Methanobacterium; the predominating genus Methanosaeta, accounting for 40.5%, 54.2%, 73.6% and 78.7% in four samples from top to bottom, indicated a major methanogenesis pathway by acetoclastic methanogenesis in the methanogenic unit. The beta diversity indexes illustrated a more similar distribution of bacterial communities than that of methanogens between acidogenic unit and methanogenic unit. The differentiation of methanogenic community composition in two phases, as well as pH values and volatile fatty acid（VFA） concentrations confirmed the phase separation of the ITPAR. Overall, the results of this study demonstrated that the special designing of ITPAR maintained a sufficient number of methanogens, more diverse communities and stronger syntrophic associations among microorganisms, which made two phase anaerobic digestion of cellulosic materials more efficient.

Abundance and diversity of ammonia-oxidizing archaea in response to various habitats in Pearl River Delta of China, a subtropical maritime zone

Ammonia-oxidizing archaea （AOA） are widely considered key to ammonia oxidation in various environments. However, little work has been conducted to simultaneously investigate the abundance and diversity of AOA as well as correlations between archaeal amoA genotypes and environmental parameters of different ecosystems at one district. To understand the abundance, diversity, and distribution of AOA in Pearl River Delta of China in response to various habitats, the archaeal amoA genes in soil, marine, river, lake, hot spring and wastewater treatment plant （WWTP） samples were investigated using real-time fluorescent quantitative PCR and clone libraries. Our analyses indicated that the diversity of AOA in various habitats was different and could be clustered into five major clades, i.e., estuary sediment, marine water/sediment, soil, hot spring and Cluster 1. Phylogenetic analyses revealed that the structure of AOA communities in similar ecological habitats exhibited strong relation. The canonical correspondence method indicated that the AOA community structure was strongly correlated to temperature, pH, total organic carbon, total nitrogen and dissolved oxygen variables. Assessing AOA amoA gene copy numbers, ranging from 6.84× 10^6 to 9.45 × 10^7 copies/g in dry soil/sediment, and 6.06× 10^6 to 2.41 ×10^7 copies/L in water samples, were higher than ammonia-oxidizing bacteria （AOB） by 1-2 orders of magnitude. However, AOA amoA copy numbers were much lower than AOB in WWTP activated sludge samples. Overall, these studies suggested that AOA may be a major contributor to ammonia oxidation in natural habitats but play a minor role in highly aerated activated sludge. The result also showed the ratio of AOA to AOB amoA gene abundance was positively correlated with temperature and less correlated with other environmental parameters. New data from our study provide increasing evidence for the relative abundance and diversity of ammonia-oxidizing archaea in the global nitrogen cycle.

Start-up of the anammox process from the conventional activated sludge in a hybrid bioreactor

The anaerobic ammonium oxidation （anammox） process was successfully started up from conventional activated sludge using a hybrid bioreactor within 2 months.The average removal efficiencies of ammonia and nitrite were both over 80%,and the maximum total nitrogen removal rate of 1.85 kg N/（m^3 ·day） was obtained on day 362 with the initial sludge concentration of 0.7 g mixed liquor suspended solids （MLSS）/L.Scanning electron microscope （SEM） observation of the granular sludge in the hybrid reactor clearly showed a high degree of compactness and cell sphericity,and the cell size was quite uniform.Transmission electron microscope photos showed that cells were round or oval,the cellular diameter was 0.6-1.0 μm,and the percentage of the anammoxosome compartment was 51%-85% of the whole cell volume.Fluorescence in situ hybridization analysis （FISH） indicated that anammox bacteria became the dominant population in the community （accounting for more than 51% of total bacteria on day 250）.Seven planctomycete 16S rRNA gene sequences were present in the 16S rRNA gene clone library generated from the biomass and affiliated to Candidatus Kuenenia stuttgartiensis and Candidatus Brocadia sp.,a new anammox species.In addition,the average effluent suspended solid （MLSS） concentrations of outlets I （above the non-woven carrier） and II （below the non-woven carrier） were 0.0009 and 0.0035 g/L,respectively.This showed that the non-woven carrier could catch the biomass effectively,which increased biomass and improved the nitrogen removal rate in the reactor.

Abundance and distribution of ammonia-oxidizing archaea in Tibetan and Yunnan plateau agricultural soils of China

As low oxygen and high ultraviolet （UV） exposure might significantly affect the microbial existence in plateau, it could lead to a specialized microbial community. To determine the abundance and distribution of ammonia-oxidizing archaea （AOA） in agricultural soil of plateau, seven soil samples were collected respectively from farmlands in Tibet and Yunnan cultivating the wheat, highland-barley, and colza, which are located at altitudes of 3200-3800 m above sea level. Quantitative PCR （q-PCR） and clone library targeting on amoA gene were used to quantify the abundances of AOA and ammonia-oxidizing bacteria （AOB）, and characterize the community structures of AOA in the samples. The number of AOA cells （9.34 × 10^7-2.32× 10^8 g^-1 soil） was 3.86-21.84 times greater than that of AOB cells （6.91 × 10^6-1.24 × 10^8 g^-1 soil） in most of the samples, except a soil sample cultivating highland- barley with an AOA/AOB ratio of 0.90. Based Kendall＇s correlation coefficient, no remarkable correlation between AOA abundance and the environmental factor was observed. Additionally, the diversities of AOA community were affected by total nitrogen and organic matter concentration in soils, suggesting that AOA was probably sensitive to several environmental factors, and could adjust its community structure to adapt to the environmental variation while maintaining its abundance.

Continuous desulfurization and bacterial community structure of an integrated bioreactor developed to treat SO_2 from a gas stream

Sulfide dioxide（SO2） is often released during the combustion processes of fossil fuels. An integrated bioreactor with two sections, namely, a suspended zone（SZ） and immobilized zone（IZ）, was applied to treat SO2 for 6 months. Sampling ports were set in both sections to investigate the performance and microbial characteristics of the integrated bioreactor. SO2 was effectively removed by the synergistic effect of the SZ and IZ, and more than 85%removal efficiency was achieved at steady state. The average elimination capacity of SO2 in the bioreactor was 2.80 g/（m3·hr） for the SZ and 1.50 g/（m3· hr） for the IZ. Most SO2 was eliminated in the SZ. The liquid level of the SZ and the water content ratio of the packing material in the IZ affected SO2 removal efficiency. The SZ served a key function not only in SO2 elimination, but also in moisture maintenance for the IZ. The desired water content in IZ could be feasibly maintained without any additional pre-humidification facilities. Clone libraries of 16 S r DNA directly amplified from the DNA of each sample were constructed and sequenced to analyze the community composition and diversity in the individual zones.The desulfurization bacteria dominated both zones. Paenibacillus sp. was present in both zones, whereas Ralstonia sp. existed only in the SZ. The transfer of SO2 to the SZ involved dissolution in the nutrient solution and biodegradation by the sulfur-oxidizing bacteria.This work presents a potential biological treatment method for waste gases containing hydrophilic compounds.

Reduction and characterization of bioaerosols in a wastewater treatment station via ventilation

Bioaerosols from wastewater treatment processes are a significant subgroup of atmospheric aerosols. In the present study,airborne microorganisms generated from a wastewater treatment station（WWTS） that uses an oxidation ditch process were diminished by ventilation.Conventional sampling and detection methods combined with cloning/sequencing techniques were applied to determine the groups,concentrations,size distributions,and species diversity of airborne microorganisms before and after ventilation. There were 3021 ± 537 CFU/m3 of airborne bacteria and 926 ± 132 CFU/m3 of airborne fungi present in the WWTS bioaerosol.Results showed that the ventilation reduced airborne microorganisms significantly compared to the air in the WWTS. Over 60% of airborne bacteria and airborne fungi could be reduced after4 hr of air exchange. The highest removal（92.1% for airborne bacteria and 89.1% for fungi） was achieved for 0.65–1.1 μm sized particles. The bioaerosol particles over 4.7 μm were also reduced effectively. Large particles tended to be lost by gravitational settling and small particles were generally carried away,which led to the relatively easy reduction of bioaerosol particles0.65–1.1 μm and over 4.7 μm in size. An obvious variation occurred in the structure of the bacterial communities when ventilation was applied to control the airborne microorganisms in enclosed spaces.