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.

Determination of the archaeal and bacterial communities in two-phase and single-stage anaerobic systems by 454 pyrosequencing

2-Phase anaerobic digestion（AD）, where the acidogenic phase was operated at 2 day hydraulic retention time（HRT） and the methanogenic phase at 10 days HRT, had been evaluated to determine if it could provide higher organic reduction and methane production than the conventional single-stage AD（also operated at 12 days HRT）. 454 pyrosequencing was performed to determine and compare the microbial communities. The acidogenic reactor of the 2-phase system yielded a unique bacterial community of the lowest richness and diversity, while bacterial profiles of the methanogenic reactor closely followed the single-stage reactor. All reactors were predominated by hydrogenotrophic methanogens, mainly Methanolinea. Unusually, the acidogenic reactor contributed up to 24%of total methane production in the 2-phase system. This could be explained by the presence of Methanosarcina and Methanobrevibacter, and their activities could also help regulate reactor alkalinity during high loading conditions through carbon dioxide production. The enrichment of hydrolytic and acidogenic Porphyromonadaceae, Prevotellaceae, Ruminococcaceae and unclassified Bacteroidetes in the acidogenic reactor would have contributed to the improved sludge volatile solids degradation, and ultimately the overall 2-phase system＇s performance. Syntrophic acetogenic microorganisms were absent in the acidogenic reactor but present in the downstream methanogenic reactor, indicating the retention of various metabolic pathways also found in a single-stage system. The determination of key microorganisms further expands our understanding of the complex biological functions in AD process.

Shifts in soil bacterial communities induced by the controlled-release fertilizer coatings

Coated controlled-release fertilizers （CRFs） have been widely applied in agriculture due to their increased efficiency. However, the widespread and a lot of coated CRFs application may leave undesired coating residues in the soil due to their slow degradation. Limited information is available on the effects of substantial residual coatings on the soil bacterial community. By adding 0, 5, 10, 20, and 50 times quantities of residual coating from conventional application amount of resin and water-soluble coated CRFs, we studied the responses of soil properties and bacterial community composition to these two residual coatings in black soil. The results showed that the resin and water-soluble coatings did not essentially alter the properties of black soil or cause dramatic changes to bacterial diversity within the test concentration range. The residual resin and water-soluble coatings also did not distinctly alter the relative abundance of the top ten bacteria at phylum level. Heatmap results suggested that the treatments were basically clustered into two groups by concentration rather than types of coating material. Pearson correlation analysis showed that the Simpson＇s diversity index of the bacterial community was significantly correlated with microbial biomass carbon （MBC, r=0.394, P〈0.05）, and the richness index abundance-based coverage estimator （ACE） of the bacterial community was significantly correlated with microbial biomass nitrogen （MBN, t=0.407, P〈0.05）. Overall, results of this study suggested that substantial residual resin and water-soluble coatings with 0-50 times quantities of residual coating from conventional application amount of coated CRFs did not generate obviously negative impacts on the bacterial community in black soil.

Wheat, maize and sunflower cropping systems selectively influence bacteria community structure and diversity in their and succeeding crop's rhizosphere

supported by the Special Fund for Agro-scientific Research in the Public Interest in China （201103001）

The Nitrogen-Cycling Network of Bacterial Symbionts in the Sponge Spheciospongia vesparium

The microbes associated with sponges play important roles in the nitrogen cycle of the coral reefs ecosystem,e.g.,nitrification,denitrification,and nitrogen fixation.However,the whole nitrogen-cycling network has remained incomplete in any individual sponge holobiont.In this study,454 pyrosequencing of the 16S rRNA genes revealed that the sponge Spheciospongia vesparium from the South China Sea has a unique bacterial community(including 12 bacterial phyla),dominated particularly by the genus Shewanella(order Alteromonadales).A total of 10 functional genes,nifH,amoA,narG,napA,nirK,norB,nosZ,ureC,nrfA,and gltB,were detected in the microbiome of the sponge S.vesparium by gene-targeted analysis,revealing an almost complete nitrogen-cycling network in this sponge.Particularly,bacterial urea utilization and the whole denitrification pathway were highlighted.MEGAN analysis suggests that Proteobacteria(e.g.,Shewanella)and Bacteroidetes(e.g.,Bizionia)are probably involved in the nitrogen cycle in the sponge S.vesparium.

Amoxicillin effects on functional microbial community and spread of antibiotic resistance genes in amoxicillin manufacture wastewater treatment system

This study aimed to reveal how amoxicillin（AMX） affected the microbial community and the spread mechanism of antibiotic resistance genes（ARGs） in the AMX manufacture wastewater treatment system. For this purpose, a 1.47 L expanded granular sludge bed（EGSB） reactor was designed and run for 241 days treating artificial AMX manufacture wastewater. 454 pyrosequencing was applied to analyze functional microorganisms in the system. The antibiotic genes OXA_（-1）, OXA_（-2）, OXA_（-10）, TEM_（-1）, CTX-M_（-1）, class I integrons（intI1） and 16 SrRNA genes were also examined in sludge samples. The results showed that the genera Ignavibacterium, Phocoenobacter,Spirochaeta, Aminobacterium and Cloacibacillus contributed to the degradation of different organic compounds（such as various sugars and amines）. And the relative quantification of eachβ-lactam resistance gene in the study was changed with the increasing of AMX concentration.Furthermore the vertical gene transfer was the main driver for the spread of ARGs rather than horizontal transfer pathways in the system.

Characterization of bacterial community dynamics in a full-scale drinking water treatment plant

Understanding the spatial and temporal dynamics of microbial communities in drinking water systems is vital to securing the microbial safety of drinking water.The objective of this study was to comprehensively characterize the dynamics of microbial biomass and bacterial communities at each step of a full-scale drinking water treatment plant in Beijing,China.Both bulk water and biofilm samples on granular activated carbon（GAC） were collected over 9 months.The proportion of cultivable cells decreased during the treatment processes,and this proportion was higher in warm season than cool season,suggesting that treatment processes and water temperature probably had considerable impact on the R2 A cultivability of total bacteria.16 s rRNA gene based 454 pyrosequencing analysis of the bacterial community revealed that Proteobacteria predominated in all samples.The GAC biofilm harbored a distinct population with a much higher relative abundance of Acidobactena than water samples.Principle coordinate analysis and one-way analysis of similarity indicated that the dynamics of the microbial communities in bulk water and biofilm samples were better explained by the treatment processes rather than by sampling time,and distinctive changes of the microbial communities in water occurred after GAC filtration.Furthermore,20 distinct OTUs contributing most to the dissimilarity among samples of different sampling locations and 6 persistent OTUs present in the entire treatment process flow were identified.Overall,our findings demonstrate the significant effects that treatment processes have on the microbial biomass and community fluctuation and provide implications for further targeted investigation on particular bacteria populations.

The molecular diversity of arbuscular mycorrhizal fungi in the arsenic mining impacted sites in Hunan Province of China

Arbuscular mycorrhizal fungi（AMF） can establish a mutualistic association with most terrestrial plants even in heavy metal contaminated environments. It has been documented that high concentrations of toxic metals, such as arsenic（As） in soil could adversely affect the diversity and function of AMF. However, there are still gaps in understanding the community composition of AMF under long-term As contaminations. In the present study, six sampling sites with different As concentrations were selected in the Realgar mining area in Hunan Province of China. The AMF biodiversity in the rhizosphere soils of the dominant plant species was investigated by sequencing the nuclear small subunit ribosomal RNA（SSU rR NA） gene fragments using 454-pyrosequencing technique. A total of 11 AMF genera were identified,namely Rhizophagus, Glomus, Funneliformis, Acaulospora, Diversispora, Claroideoglomus, Scutellopora,Gigaspora, Ambispora, Praglomus, and Archaeospora, among which Glomus, Rhizophagus, and Claroideoglomus clarodeum were detected in all sampling sites, and Glomus was the dominant AMF genus in the Realgar mining area. Redundancy analysis indicated that soil pH, total As and Cd concentrations were the main factors influencing AMF community structure. There was a negative correlation between the AMF species richness and the total As concentration in the soil,but no significant correlation between the Shannon–Wiener index of the AMF and plants. Our study showed that high As concentrations can exert a selective effect on the AMF populations.

Temporal and spatial changes of microbial community in an industrial effluent receiving area in Hangzhou Bay

Anthropogenic activities usually contaminate water environments, and have led to the eutrophication of many estuaries and shifts in microbial communities. In this study, the temporal and spatial changes of the microbial community in an industrial effluent receiving area in Hangzhou Bay were investigated by 454 pyrosequencing. The bacterial community showed higher richness and biodiversity than the archaeal community in all sediments. Proteobacteria dominated in the bacterial communities of all the samples; MarineGroupⅠand Methanomicrobia were the two dominant archaeal classes in the effluent receiving area. PCoA and AMOVA revealed strong seasonal but minor spatial changes in both bacterial and archaeal communities in the sediments. The seasonal changes of the bacterial community were less significant than those of the archaeal community, which mainly consisted of fluctuations in abundance of a large proportion of longstanding species rather than the appearance and disappearance of major archaeal species. Temperature was found to positively correlate with the dominant bacteria, Betaproteobacteria, and negatively correlate with the dominant archaea,MarineGroupⅠ; and might be the primary driving force for the seasonal variation of the microbial community.

Direct evidence of microbiological water quality changes on bacterial quantity and community caused by plumbing system

Drinking water quality deteriorates from treatment plant to customer taps, especially in the plumbing system. There is no direct evidence about what the differences are contributed by plumbing system. This study compared the water quality in the water main and at customer tap by preparing a sampling tap on the water main. The biomass was quantified by adenosine triphosphate(ATP) and the microbial community was profiled by 454 pyrosequencing.The results showed that in distribution pipes, biofilm contributed >94% of the total biomass,while loose deposits showed little contribution(< 2%) because of the low amount of loose deposits. The distribution of biological stable water had minor effects on the microbiocidal water quality regarding both quantity(ATP 1 ng/L vs. 1.7 ng/L) and community of the bacteria. Whereas the plumbing system has significant contribution to the increase of active biomass(1.7 ng/L vs. 2.9 ng/L) and the changes of bacterial community. The relative abundance of Sphingomonas spp. at tap(22%) was higher than that at water main(2%), while the relative abundance of Pseudomonas spp. in tap water(15%) was lower than that in the water from street water main(29%). Though only one location was prepared and studied, the present study showed that the protocol of making sampling tap on water main offered directly evidences about the impacts of plumbing system on tap water quality, which makes it possible to distinguish and study the processes in distribution system and plumbing system separately.

Contrasting soil fungal communities in Mediterranean pine forests subjected to different wildfire frequencies

Mediterranean forest ecosystems are characterized by various vascular plant groups with their associated mycor-rhizae and free living soil fungi with various ecological functions.Fire plays a major role in Mediterranean ecosystem dynamics and impacts both above-and below-ground community structure and functioning.However,studies on the effects induced by altered disturbance regimes(associated with recent land use and climate extremes)on fire ecology and especially on its below-ground impacts are few.The objectives of this study were to evaluate the effects of different wildfire regimes on soil fungal community structure using two different molecular methods.We investigated the long-term effects of wildfire on soil fungal communities associated with Pinus pinaster forests in central Portugal,by comparing the results of denaturing gradient gel electrophoresis(DGGE)-based profiling with those obtained with 454 pyrosequencing.Four forest stands with differing fire history and fire return interval,and vegetation cover(mature forest,early successional stage of pine regeneration,and forest converted to scrubland)were sampled 6 years after the last fire event.The pyrosequencing-based approach indicated ca.eight-fold higher numbers of taxa than DGGE.However,fungal community fingerprinting data obtained for the different study stands with DGGE were congruent with those obtained with pyrosequencing.Both short(7.6 years)and long(24 years)fire return intervals(indicated by the presence of ericaceous shrubs in the understorey)induced a decrease in the abun-dance ratio between basidiomycetes and ascomycetes and appeared to reduce the frequency of ectomycorrhizal fungal species and saprophytes.Wildfire significantly reduced the frequency of late stage successional taxa(e.g.Atheliaceae and Cantharellales)and known or putative saprophytes belonging to the Clavulinaceae and the Archaeorhizomycetaceae.Conversely,early successional fungal species belonging to the Thelephoraceae were favoured by both fire return inter-vals,while the abundance of Cortinarius and Hebeloma,which include several Cistus-specific species,increased with short wildfire return intervals.This last finding highlights the relationship between postfire vegetation composition and cover(vegetation successional stage),and fungal symbionts.We hypothesise that these changes could,in the long term,exhaust the resilience of Mediterranean pine forest vegetation and associated soil fungal communities by preventing pine regeneration.

A conserved arbuscular mycorrhizal fungal core-species community colonizes potato roots in the Andes

Plant-symbiotic arbuscular mycorrhizal fungi(AMF)are of high global ecological and economic importance,but describing environmental communities of AMF at the species level remains a challenge,despite the need to understand AMF-plant preferences and to apply AMF in sustainable agriculture.Here,the potato-associated AMF species community composition was assessed for three Andean countries along an altitudinal gradient and at different plant stages,by using 454 GS-FLX+sequencing of a 760 bp LSU rRNA gene PCR amplicon.Two methods were compared:defining OTUs based on a simple sequence similarity threshold,or affiliating reference sequences to species based on a high throughput phylogenetic annotation approach using an evolutionary placement algorithm(EPA).The EPA-based approach was not only more precise,but also fundamental to robustly unveil the AMF species community composition.The principal advantage of this approach was also demonstrated by using artificially constructed datasets based on validated public database sequences.The affiliation of sequence reads to species using phylogenetic annotation revealed a surprisingly conserved AMF core-species community structure in Andean potatoes,regardless of different plant stages and environmental factors.In total,41 species were detected and in some cases more than 25 species were found colonizing an individual root system.Acaulospora species were identified as dominant colonizers,co-occurring with Cetraspora nodosa and certain Claroideoglomus and Rhizophagus species in most potato root samples.