厌氧颗粒污泥微生物群落结构与功能的研究进展

厌氧颗粒污泥(anaerobicgranularsludge,AnGS)是由多种功能微生物组成的自固定化聚集体,具有容积负荷高、工艺简单、剩余污泥产量低等优点,在废水处理领域中显示出巨大的技术和经济潜力,被认为是一种很有前景的低碳废水处理工艺。本文系统总结了近年来厌氧颗粒污泥微生物结构和功能的研究成果,从微生物学角度讨论了厌氧颗粒污泥形成及稳定的影响因素,并对今后厌氧颗粒污泥的研究进行了展望,以期为后续厌氧颗粒污泥技术的深入研究和实际工程应用提供参考。     

Effects of ferrous iron on the performance and microbial community in aerobic granular sludge in relation to nutrient removal

Lab‐scale experiments were conducted to investigate the effects of ferrous iron on nutrient removal performance and variations in the microbial community inside aerobic granular sludge for 408 days. Two reactors were simultaneously operated, one without added ferrous iron (SBR1), and one with 10 mg Fe²⁺ L^?1 of added ferrous iron (SBR2). A total of 1 mg Fe²⁺ L^?1 of added ferrous iron was applied to SBR1 starting from the 191st day to observe the resulting variations in the nutrient removal performance and the microbial community. The results show that ammonia‐oxidizing bacteria (AOB) could not oxidize ammonia due to a lack of iron compounds, but they could survive in the aerobic granular sludge. Limited ferrous iron addition encouraged nitrification. Enhanced biological phosphorus removal (EBPR) from both reactors could not be maintained regardless of the amount of ferrous iron that was applied. EBPR was established in both reactors when the concentration of mixed liquor suspended solid (MLSS) and the percentage of Accumulibacteria increased. A total of 10 mg Fe²⁺ L^?1 of added ferrous iron had a relatively adverse effect on the growth of AOB species compared to 1 mg Fe²⁺ L^?1 of added ferrous iron, but it encouraged the growth of Nitrospira sp. and Accumulibacteria, which requires further study. It could be said that the compact and stable structure of aerobic granular sludge preserved AOB and NOB from Fe‐deficient conditions, and wash‐out during the disintegration period. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:716–725, 2017     

Discovery and characterization of a new bacterial candidate division by an anaerobic sludge digester metagenomic approach

We have constructed a large fosmid library from a mesophilic anaerobic digester and explored its 16S rDNA diversity using a high-density filter DNA–DNA hybridization procedure. We identified a group of 16S rDNA sequences forming a new bacterial lineage named WWE3 (Waste Water of Evry 3). Only one sequence from the public databases shares a sequence identity above 80% with the WWE3 group which hence cannot be affiliated to any known or candidate prokaryotic division. Despite representing a non-negligible fraction (5% of the 16S rDNA sequences) of the bacterial population of this digester, the WWE3 bacteria could not have been retrieved using the conventional 16S rDNA amplification procedure due to their unusual 16S rDNA gene sequence. WWE3 bacteria were detected by polymerase chain reaction (PCR) in various environments (anaerobic digesters, swine lagoon slurries and freshwater biofilms) using newly designed specific PCR primer sets. Fluorescence in situ hybridization (FISH) analysis of sludge samples showed that WWE3 microorganisms are oval-shaped and located deep inside sludge flocs. Detailed phylogenetic analysis showed that WWE3 bacteria form a distinct monophyletic group deeply branching apart from all known bacterial divisions. A new bacterial candidate division status is proposed for this group.     

Acidification of methanol-fed anaerobic granular sludge bioreactors by cobalt deprivation: Induction and microbial community dynamics

The acidification of mesophilic (30 degrees C) methanol-fed upflow anaerobic sludge bed (UASB) reactors induced by cobalt deprivation from the influent was investigated by coupling the reactor performance (pH 7.0; organic loading rate 4.5 g COD . L(-1) . d(-1)) to the microbial ecology of the bioreactor sludge. The latter was investigated by specific methanogenic activity (SMA) measurements and fluorescence in situ hybridization (FISH) to quantify the abundance of key organisms over time. This study hypothesized that under cobalt limiting conditions, the SMA on methanol of the sludge gradually decreases, which ultimately results in methanol accumulation in the reactor effluent. Once the methanol accumulation surpasses a threshold value (about 8.5 mM for the sludge investigated), reactor acidification occurs because acetogens outcompete methylothrophic methanogens at these elevated methanol concentrations. Methanogens present in granular sludge at the time of the acidification do not use methanol as the direct substrate and are unable to degrade acetate. Methylotrophic/acetoclastic methanogenic activity was found to be lost within 10 days of reactor operation, coinciding with the disappearance of the Methanosarcina population. The loss of SMA on methanol can thus be used as an accurate parameter to predict reactor acidification of methanol-fed UASB reactors operating under cobalt limiting conditions.     

A metagenomic approach to characterization of the vaginal microbiome signature in pregnancy

While current major national research efforts (i.e., the NIH Human Microbiome Project) will enable comprehensive metagenomic characterization of the adult human microbiota, how and when these diverse microbial communities take up residence in the host and during reproductive life are unexplored at a population level. Because microbial abundance and diversity might differ in pregnancy, we sought to generate comparative metagenomic signatures across gestational age strata. DNA was isolated from the vagina (introitus, posterior fornix, midvagina) and the V5V3 region of bacterial 16S rRNA genes were sequenced (454FLX Titanium platform). Sixty-eight samples from 24 healthy gravidae (18 to 40 confirmed weeks) were compared with 301 non-pregnant controls (60 subjects). Generated sequence data were quality filtered, taxonomically binned, normalized, and organized by phylogeny and into operational taxonomic units (OTU); principal coordinates analysis (PCoA) of the resultant beta diversity measures were used for visualization and analysis in association with sample clinical metadata. Altogether, 1.4 gigabytes of data containing >2.5 million reads (averaging 6,837 sequences/sample of 493 nt in length) were generated for computational analyses. Although gravidae were not excluded by virtue of a posterior fornix pH >4.5 at the time of screening, unique vaginal microbiome signature encompassing several specific OTUs and higher-level clades was nevertheless observed and confirmed using a combination of phylogenetic, non-phylogenetic, supervised, and unsupervised approaches. Both overall diversity and richness were reduced in pregnancy, with dominance of Lactobacillus species (L. iners crispatus, jensenii and johnsonii, and the orders Lactobacillales (and Lactobacillaceae family), Clostridiales, Bacteroidales, and Actinomycetales. This intergroup comparison using rigorous standardized sampling protocols and analytical methodologies provides robust initial evidence that the vaginal microbial 16S rRNA gene catalogue uniquely differs in pregnancy, with variance of taxa across vaginal subsite and gestational age.     

Long-term effects of multi-walled carbon nanotubes on the performance and microbial community structures of an anaerobic granular sludge system

Multi-walled carbon nanotubes (MWCNTs) released into the sewage may cause negative and/or positive effects on the treatment system. The objective of this study was to explore over 110 days’ effect of MWCNTs on the performance of anaerobic granular sludge and microbial community structures in an upflow anaerobic sludge blanket (UASB) reactor. The results showed that MWCNTs had no significant effect on the removal of chemical oxidation demand (COD) and ammonia in UASB reactor, but the total phosphorus (TP) removal efficiency increased by 29.34%. The biogas production of the reactor did not change. The anaerobic granular sludge tended to excrete more EPS to resist the effects of MWCNTs during the long-term impact. Illumina MiSeq sequencing of 16S rRNA gene revealed that MWCNTs did not affect the microbial diversity, but altered the composition and structure of microbial community in the reactor. In this process, Saccharibacteria replaced Proteobacteria as the highest abundant bacterial phylum. MWCNTs promoted the differentiation of methanogen structure, resulting in increase of Methanomassiliicoccus, Methanoculleus, and the uncultured WCHA1–57. These results indicated that MWCNTs impacted the performance of UASB reactor and the structures of the microbial community in anaerobic granular sludge.

     

The effect of long-term mercury pollution on the soil microbial community

The effect of long-term exposure to mercury on the soil microbial community was investigated in soil from three different sites along a pollution gradient. The amount of total and bioavailable mercury was negatively correlated to the distance from the center of contamination. The size of the bacterial and protozoan populations was reduced in the most contaminated soil, whereas there was no significant difference in fungal biomass measured as chitinase activity. Based on the number of colony morphotypes, moreover, the culturable bacterial population was structurally less diverse and contained a higher proportion of resistant and fast-growing forms. The profiles of amplified 16S rDNA sequences obtained from community DNA by denaturating gradient gel electrophoresis (DGGE) also reflected the altered community structure and decreased diversity along the mercury gradient as expressed in terms of the number and abundance of bands. The functional potential of the microbial population measured as sole carbon source utilization by Ecoplates((R)) differed between the soils, but there was no change in the number of substrates utilized. The observed changes in the different soil microbial populations are probably a combination of both direct and indirect effects of the mercury contamination.     

Effective anaerobic treatment of fresh leachate from MSW incineration plant and dynamic characteristics of microbial community in granular sludge

We investigated the treatment of fresh leachate from municipal solid waste incineration plants with high-strength organics using a lab-scale expanded granular sludge bed (EGSB) reactor. The reactor was operated at a mesophilic temperature (33 °C) for 118 days. The influent chemical oxygen demand (COD) of the leachate gradually increased to over 70,000 mg/L, and the organic loading rate increased to 18 kg COD/(m³?day). An average COD removal efficiency of 86.7 % was achieved when the reactor was fed with raw leachate, which suggests the feasibility of the EGSB process for leachate treatment. The microbial communities in the sludge from the reactor during the trial operation were constructed by denaturing gradient gel electrophoresis, clone libraries, and real-time quantitative polymerase chain reaction. The dominant group for archaea was Methanosaeta, with 68.4 % proportion at the start of the operation, and then changed to Methanosarcina, with a proportion of 62.3 %, after 118 days of operation. The dominant group of eubacteria was confirmed to be Firmicutes throughout the operation process, with the proportion increasing from >50 to 81.2 %. Almost all the operational taxonomic units of Firmicutes belonged to the order Clostridiales, with characteristic spore formation. The microbial diversity of the population was low under raw leachate as feed in the reactor. The dynamics of the microbial community in the anaerobic granular sludge was discussed relating with the operating status of the EGSB reactor.     

Leaching of pyrite-arsenopyrite concentrate in bioreactors during continuous cultivation of a thermoacidophilic microbial community

A community of thermoacidophilic chemolithotrophic microorganisms was shown to exhibit enhanced efficiency of leaching and biooxidation of the gold-bearing pyrite-arsenopyrite flotation concentrate in continuous mode of cultivation. Under the optimal values of growth parameters, the degree of oxidation of sulfide arsenic, iron, sulfur, and antimony in the line of three laboratory reactors (D = 0.004 h^?1) was 99.55, 98.87, 99.65, and 97.08%, respectively, while gold recovery from the solid biooxidation residue was 97.4%.     

Quantitative analysis of a high-rate hydrogen-producing microbial community in anaerobic agitated granular sludge bed bioreactors using glucose as substrate

Fermentative H₂ production microbial structure in an agitated granular sludge bed bioreactor was analyzed using fluorescence in situ hybridization (FISH) and polymerase chain reaction-denatured gradient gel electrophoresis (PCR-DGGE). This hydrogen-producing system was operated at four different hydraulic retention times (HRTs) of 4, 2, 1, and 0.5 h and with an influent glucose concentration of 20 g chemical oxygen demand/l. According to the PCR-DGGE analysis, bacterial community structures were mainly composed of Clostridium sp. (possibly Clostridium pasteurianum), Klebsiella oxytoca, and Streptococcus sp. Significant increase of Clostridium/total cell ratio (68%) was observed when the reactor was operated under higher influent flow rate. The existence of Streptococcus sp. in the reactor became more important when operated under a short HRT as indicated by the ratio of Streptococcus probe-positive cells to Clostridium probe-positive cells changing from 21% (HRT 4 h) to 38% (HRT 0.5 h). FISH images suggested that Streptococcus cells probably acted as seeds for self-flocculated granule formation. Furthermore, combining the inspections with hydrogen production under different HRTs and their corresponding FISH analysis indicated that K. oxytoca did not directly contribute to H₂ production but possibly played a role in consuming O₂ to create an anaerobic environment for the hydrogen-producing Clostridium.     

COD removal from expanded granular sludge bed effluent using a moving bed biofilm reactor and their microbial community analysis

The bioreactor performance of a moving bed biofilm reactor (MBBR) as post-treatment of expanded granular sludge bed (EGSB) effluent was investigated. Moreover, the microbial communities of the two bioreactors during different operation periods were studied. The MBBR was efficient for COD removal with the mean efficiency of 82.4%, and produced an effluent with high and stable quality against shock loading resulting from the low temperature applied to EGSB. The study indicates that the microbial community in the reactors could adapt to perturbations such as influent wastewater characteristics and operation temperature, which is beneficial to maintain efficient and stable COD removal in the combined EGSB-MBBR system. Archaeal 16S rRNA gene sequence analysis indicated the presence of Methanomethylovorans, Methanolinea, Methanoregula boonei, Methanosarcina barkeri, and Methanospirillum hungatei in the EGSB. Bacterial 16S rRNA gene sequence analysis indicated the presence of Runella limosa, Dokdonella, Sphaerotilus, Hydrogenophaga, and Pseudomonas in the MBBR. The EGSB-MBBR system established here could be used as an efficient option for organic matter removal, which holds a great potential in practical applications for nutrients (N and P) removal.     

The microbial community composition of a nitrifying-denitrifying activated sludge from an industrial sewage treatment plant analyzed by the full-cycle rRNA approach

The composition of the microbial community present in the nitrifying-denitrifying activated sludge of an industrial wastewater treatment plant connected to a rendering facility was investigated by the full-cycle rRNA approach. After DNA extraction using three different methods, 94 almost full-length 16S rRNA gene clones were retrieved and analyzed phylogenetically. 59% of the clones were affiliated with the Proteobacteria and clustered with the beta- (29 clones), alpha- (24), and delta-class (2 clones), respectively. 15 clones grouped within the green nonsulfur (GNS) bacteria and 11 clones belonged to the Planctomycetes. The Verrucomicrobia, Acidobacteria, Nitrospira, Bacteroidetes, Firmicutes and Actinobacteria were each represented by one to five clones. Interestingly, the highest 'species richness' [measured as number of operational taxonomic units (OTUs)] was found within the alpha-class of Proteobacteria, followed by the Planctomycetes, the beta-class of Proteobacteria, and the GNS-bacteria. The microbial community composition of the activated sludge was determined quantitatively by using 36 group-, subgroup-, and OTU-specific rRNA-targeted oligonucleotide probes for fluorescence in situ hybridization (FISH), confocal laser scanning microscopy and digital image analysis. 89% of all bacteria detectable by FISH with a bacterial probe set could be assigned to specific divisions. Consistent with the 16S rRNA gene library data, members of the beta-class of Proteobacteria dominated the microbial community and represented almost half of the biovolume of all bacteria detectable by FISH. Within the beta-class, 98% of the cells could be identified by the application of genus- or OTU-specific probes demonstrating a high in situ abundance of bacteria related to Zoogloea and Azoarcus sensu lato. Taken together, this study provides the first encompassing, high-resolution insight into the in situ composition of the microbial community present in a full-scale, industrial wastewater treatment plant.     

Molecular characterization of mesophilic and thermophilic sulfate reducing microbial communities in expanded granular sludge bed (EGSB) reactors

The microbial communities established in mesophilic and thermophilic expanded granular sludge bed reactors operated with sulfate as the electron acceptor were analyzed using 16S rRNA targeted molecular methods, including denaturing gradient gel electrophoresis, cloning, and phylogenetic analysis. Bacterial and archaeal communities were examined over 450 days of operation treating ethanol (thermophilic reactor) or ethanol and later a simulated semiconductor manufacturing wastewater containing citrate, isopropanol, and polyethylene glycol 300 (mesophilic reactor), with and without the addition of copper(II). Analysis, of PCR-amplified 16S rRNA gene fragments using denaturing gradient gel electrophoresis revealed a defined shift in microbial diversity in both reactors following a change in substrate composition (mesophilic reactor) and in temperature of operation from 30°C to 55°C (thermophilic reactor). The addition of copper(II) to the influent of both reactors did not noticeably affect the composition of the bacterial or archaeal communities, which is in agreement with the very low soluble copper concentrations (3–310 μg l⁻¹) present in the reactor contents as a consequence of extensive precipitation of copper with biogenic sulfides. Furthermore, clone library analysis confirmed the phylogenetic diversity of sulfate-reducing consortia in mesophilic and thermophilic sulfidogenic reactors operated with simple substrates.     

Formation of poly-3-hydroxybutyrate by a soil microbial community during batch and heterocontinuous cultivation

Glucose added to soil as an extracellular source of carbon and energy was proved to be deposited into an intracellular polymer poly-3-hydroxybutyrate (PHB). Untreated soil samples contained PHB amounts corresponding to 1.56 –2.64 μg of crotonic acid per 1 g soil. During batch cultivation after addition of 1 % glucose the PHB content increased by 20-fold after 2 d and then decreased owing to the disappearance of glucose from the soil. Repeated additions of glucose did not bring about any significant increase in PHB content as compared with a single addition. In soil supplied continuously with 0.1 or 0.25 % glucose solution, the content of PHB increased, after an initial lag, gradually up to the 10th day. After 1-d cultivation the content of PHB in the batch system increased even in the presence of diammonium hydrogen phosphate. In a heterocontinuous system no PHB accumulation took place in the presence of this source of nitrogen and phosphorus as long as the C:N ratio of theadded substrate was 10: 1.     

Effect of dilution rate on the microbial structure of a mesophilic butyrate-degrading methanogenic community during continuous cultivation

We constructed two mesophilic anaerobic chemostats that were continuously fed with synthetic wastewater containing butyrate as the sole source of carbon and energy. Steady-state conditions were achieved at dilution rates between 0.025 and 0.7 day⁻¹. Butyrate, fed into the chemostat, was almost completely mineralized to CH₄ and CO₂ at dilution rates below 0.5 day⁻¹. The butyrate-degrading methanogenic communities in the chemostats at dilution rates between 0.025 and 0.7 day⁻¹ were monitored based on the 16S rRNA gene, using molecular biological techniques including clone library analysis, denaturing gradient gel electrophoresis, and quantitative real-time polymerase chain reaction. The aceticlastic methanogen Methanosaeta and the hydrogenotrophic methanogen Methanoculleus dominated in methanogens at low dilution rates, whereas the aceticlastic methanogen Methanosaeta, Methanosarcina, the hydrogenotrophic methanogen Methanoculleus, and Methanospirillum dominated at high dilution rates. Bacteria affiliated with the family Syntrophaceae in the phylum Proteobacteria predominated at the low dilution rate of 0.025 day⁻¹, whereas bacteria affiliated with the phylum Firmicutes and Candidate division OP3 predominated at high dilution rates. A significant quantity of bacteria closely related to the genus Syntrophomonas was detected at high dilution rates. Dilution rate showed an apparent effect on archaeal and bacterial communities in the butyrate-fed chemostats.     

Effect of seed sludge microbial community and activity on the performance of anaerobic reactors during the start-up period

In this study, two laboratory-scale anaerobic batch reactors started up with different inoculum sludges and fed with the same synthetic wastewater were monitored in terms of performance and microbial community shift by denaturant gradient gel electrophoresis fingerprinting and subsequent cloning, sequencing analysis in order to reveal importance of initial quality of inoculum sludge for operation of anaerobic reactors. For this purpose, two different seed sludge were evaluated. In Reactor1 seeded with a sludge having less diverse microbial community (19 operational taxonomic unit (OTU’s) for Bacterial and 8 OTU’s for Archaeal community, respectively) and a methanogenic activity of 150 ml CH₄ g TVS⁻¹ day⁻¹, a chemical oxygen demand (COD) removal efficiency of 78.8 ± 4.17% was obtained at a substrate to microorganism (S/X) ratio of 0.38. On the other hand, Reactor2, seeded with a sludge having a much more diverse microbial community (24 OTU’s for Bacterial and 9 OTU’s for Archaeal communities, respectively) and a methanogenic activity, 450 ml CH₄ g TVS⁻¹ day⁻¹, operated in the same conditions showed a better start-up performance; a COD removal efficiency of over 98% at a S/X ratio of 0.53. Sequence analysis of Seed2 revealed the presence of diverse fermentative and syntrophic bacteria, whereas excised bands of Seed1 related to fermentative and sulfate/metal-reducing bacteria. This study revealed that a higher degree of bacterial diversity, especially the presence of syntrophic bacteria besides the abundance of key species such as methanogenic Archaea may play an important role in the performance of anaerobic reactors during the start-up period.     

The Sorcerer II Global Ocean Sampling Expedition: metagenomic characterization of viruses within aquatic microbial samples

Viruses are the most abundant biological entities on our planet. Interactions between viruses and their hosts impact several important biological processes in the world's oceans such as horizontal gene transfer, microbial diversity and biogeochemical cycling. Interrogation of microbial metagenomic sequence data collected as part of the Sorcerer II Global Ocean Expedition (GOS) revealed a high abundance of viral sequences, representing approximately 3% of the total predicted proteins. Cluster analyses of the viral sequences revealed hundreds to thousands of viral genes encoding various metabolic and cellular functions. Quantitative analyses of viral genes of host origin performed on the viral fraction of aquatic samples confirmed the viral nature of these sequences and suggested that significant portions of aquatic viral communities behave as reservoirs of such genetic material. Distributional and phylogenetic analyses of these host-derived viral sequences also suggested that viral acquisition of environmentally relevant genes of host origin is a more abundant and widespread phenomenon than previously appreciated. The predominant viral sequences identified within microbial fractions originated from tailed bacteriophages and exhibited varying global distributions according to viral family. Recruitment of GOS viral sequence fragments against 27 complete aquatic viral genomes revealed that only one reference bacteriophage genome was highly abundant and was closely related, but not identical, to the cyanomyovirus P-SSM4. The co-distribution across all sampling sites of P-SSM4-like sequences with the dominant ecotype of its host, Prochlorococcus supports the classification of the viral sequences as P-SSM4-like and suggests that this virus may influence the abundance, distribution and diversity of one of the most dominant components of picophytoplankton in oligotrophic oceans. In summary, the abundance and broad geographical distribution of viral sequences within microbial fractions, the prevalence of genes among viral sequences that encode microbial physiological function and their distinct phylogenetic distribution lend strong support to the notion that viral-mediated gene acquisition is a common and ongoing mechanism for generating microbial diversity in the marine environment.