Characterization of the Microbial Community in a Partial Nitrifying Sequencing Batch Biofilm Reactor

A lab-scale partial nitrifying sequencing batch biofilm reactor was a successful start-up. Denaturing gradient gel electrophoresis (DGGE) was used to investigate the bacterial community dynamics in three periods together with inocula sludge at ambient temperature. The DGGE profiles of bacteria and Shannon–Wiener index (H′) results showed that high free ammonia (FA) concentration referred to lower diversity in the bioreactor system. Cluster analysis indicated that microorganism in period III was similar with inocula sludge and was different from that in periods I and II. Similar results also appeared in ammonia-oxidizing bacteria (AOB) community structure and nitrite-oxidizing bacteria (NOB) community structure, and at least four AOB species and two NOB species were present in period III, respectively. Phylogenetic analysis of amoA gene sequences showed that Nitrosomonas eutropha cluster was predominant in all the three periods. With lower ammonium loads, three new operational taxonomic units formed and consisted Nitrosomonas sp. Cluster. This article demonstrated that microbial community, AOB, and NOB diversity were related with FA concentration closely at ambient temperature.     

Bacterial Diversity and Function of Aerobic Granules Engineered in a Sequencing Batch Reactor for Phenol Degradation

Aerobic granules are self-immobilized aggregates of microorganisms and represent a relatively new form of cell immobilization developed for biological wastewater treatment. In this study, both culture-based and culture-independent techniques were used to investigate the bacterial diversity and function in aerobic phenol- degrading granules cultivated in a sequencing batch reactor. Denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rRNA genes demonstrated a major shift in the microbial community as the seed sludge developed into granules. Culture isolation and DGGE assays confirmed the dominance of β-Proteobacteria and high-G+C gram-positive bacteria in the phenol-degrading aerobic granules. Of the 10 phenol-degrading bacterial strains isolated from the granules, strains PG-01, PG-02, and PG-08 possessed 16S rRNA gene sequences that matched the partial sequences of dominant bands in the DGGE fingerprint belonging to the aerobic granules. The numerical dominance of strain PG-01 was confirmed by isolation, DGGE, and in situ hybridization with a strain-specific probe, and key physiological traits possessed by PG-01 that allowed it to outcompete and dominate other microorganisms within the granules were then identified. This strain could be regarded as a functionally dominant strain and may have contributed significantly to phenol degradation in the granules. On the other hand, strain PG-08 had low specific growth rate and low phenol degradation ability but showed a high propensity to autoaggregate. By analyzing the roles played by these two isolates within the aerobic granules, a functional model of the microbial community within the aerobic granules was proposed. This model has important implications for rationalizing the engineering of ecological systems.     

Impact of an Aerobic Thermophilic Sequencing Batch Reactor on Antibiotic-Resistant Anaerobic Bacteria in Swine Waste

The introduction of antibiotics to animal feed has contributed to the selection of antibiotic-resistant bacteria in concentrated animal feeding operations. The aim of this work was to characterize the impact of an aerobic thermophilic biotreatment on anaerobic antibiotic-resistant bacteria in swine waste. Despite 162- to 6,166-fold reduction in antibiotic-resistant populations enumerated in the swine waste at 25°C and 37°C, resistant populations remained significant (10⁴ to 10⁵ most probable number per milliliter) in the treated swine waste. Five resistance genes were detected before [tet(LMOS) erm(B)], and six resistance genes were detected after [tet(LMOSY) erm(B)] biotreatment. However, the biotreatment decreased the frequency of detection of resistance genes by 57%. Analysis by denaturing gradient gel electrophoresis of polymerase chain reaction-amplified 16 S ribosomal DNA (rDNA) fragments showed that the biotreatment reduced the bacterial diversity of resistant populations enumerated at 37°C. Cloning and sequencing of the 16 S rDNA of these populations revealed that most clones in the treated swine waste were closely similar to some of the clones retrieved from the untreated swine waste. This study revealed that the aerobic thermophilic biotreatment developed in our laboratory does not prevent the introduction of facultatively anaerobic antibiotic-resistant bacteria and their resistance genes into agricultural ecosystems. Horizontal transfer of ecologically advantageous genes within microbial communities are likely to prevent thermophilic biotreatments from completely eliminating antibiotic-resistant bacteria and their resistance genes in animal wastes.     

Fate of Chlortetracycline- and Tylosin-Resistant Bacteria in an Aerobic Thermophilic Sequencing Batch Reactor Treating Swine Waste

Antibiotics have been added to animal feed for decades. Consequently, food animals and their wastes constitute a reservoir of antibiotic-resistant bacteria. The objective of this work was to characterize the impact of an aerobic thermophilic biotreatment on aerobic, antibiotic-resistant bacteria in swine waste. The proportion of tylosin- and chlortetracycline-resistant bacteria grown at 25°C, 37°C, and 60°C decreased after treatment, but they were still abundant (10² to 10⁸ most probable number ml⁻¹) in the treated swine waste. The presence of 14 genes conferring resistance to tylosin and chlortetracycline was assessed by polymerase chain reaction in bacterial populations grown at 25°C, 37°C, and 60°C, with or without antibiotics. In 22 cases, genes were detected before but not after treatment. The overall gene diversity was wider before [tet(BLMOSY), erm(AB)] than after [tet(LMOS), erm(B)] treatment. Analysis by denaturing gradient gel electrophoresis of amplified 16S ribosomal DNA (rDNA) fragments generally showed a reduction of the bacterial diversity, except for total populations grown at 60°C and for tylosin-resistant populations grown at 37°C. The latter were further investigated by cloning and sequencing their 16S rDNA. Phylotypes found before treatment were all closely related to Enterococcus hirae, whereas six different phylotypes, related to Pseudomonas, Alcaligenes, and Pusillimonas, were found after treatment. This work demonstrated that the aerobic thermophilic biotreatment cannot be considered as a means for preventing the dissemination of aerobic antibiotic-resistant bacteria and their resistance genes to the environment. However, since pathogens do not survive the biotreatment, the effluent does not represent an immediate threat to animal or human health.     

Analysis of Microbial Community during Biofilm Development in an Anaerobic Wastewater Treatment Reactor

The formation, structure, and biodiversity of a multispecies anaerobic biofilm inside an Upflow Anaerobic Sludge Bed (UASB) reactor fed with brewery wastewater was examined using complementary microbial ecology methods such us fluorescence in situ hybridization (FISH), denaturing gradient gel electrophoresis (DGGE), and cloning. The biofilm development can be roughly divided into three stages: an initial attachment phase (0-36 h) characterized by random adhesion of the cells to the surface; a consolidation phase (from 36 h to 2 weeks) defined by the appearance of microcolonies; and maturation phase (from 2 weeks to 2 months). During the consolidation period, proteobacteria with broad metabolic capabilities, mainly represented by members of alpha-Proteobacteria class (Oleomonas, Azospirillum), predominated. Beta-, gamma-, delta- (both syntrophobacteria and sulfate-reducing bacteria) and epsilon- (Arcobacter sp.) Proteobacteria were also noticeable. Archaea first appeared during the consolidation period. A Methanospirillum-like methanogen was detected after 36 h, and this was followed by the detection of Methanosarcina, after 4 days of biofilm development. The mature biofilm displayed a hill and valley topography with cells embedded in a matrix of exopolymers where the spatial distribution of the microorganisms became well-established. Compared to the earlier phases, the biodiversity had greatly increased. Although alpha-Proteobacteria remained as predominant, members of the phyla Firmicutes, Bacteroidete, and Thermotogae were also detected. Within the domain Archaea, the acetoclastic methanogen Methanosaeta concilii become dominant. This study provides insights on the trophic web and the shifts in population during biofilm development in an UASB reactor.     

454测序技术在微生物生态学研究中的应用

以Sanger法(双脱氧核苷酸末端终止法)为代表的第1代测序技术由于其成本高、速度慢、通量低等不足,满足不了大规模测序的要求.进入21世纪后,以Roche 454为代表的第2代测序技术诞生了,454测序法作为一种高通量的测序方法,近年来已被广泛应用于微生物生态学研究中.介绍了该测序技术的原理和操作步骤,结合本实验室的研...     

Aerobic Biodegradation of Mononitrotoluenes in Batch and Continuous Reactor Systems

Degradations of nitrotoluenes, individually and in a mixture, were carried out in batch and continuous aerobic reactors by a defined mixed microbial culture. The degradation rates and efficiencies of the isomers were evaluated in batch and continuous reactors. The results demonstrated that all the three-mononitrotoluene isomers were degraded simultaneously and completely in presence of excess oxygen. The nitro group position on the benzene ring influenced the degradation rates of the individual MNT isomers in batch systems. In the continuous biofilm reactor with a sufficient biocatalyst quantity, quality and excessive oxygen supply rate, the degradation rates of the mononitrotoluenes were almost identical as long as the compounds were present individually and their loading did not exceed the capacity of the catabolic master reaction. The microbial composition of the biofilm changed qualitatively and quantitatively during long-term continuous operation under aerobic non-aseptic conditions. This complex investigation resulted in data that can be applied for the scale-up procedure for field experiments.     

454测序法在环境微生物生态研究中的应用

传统的Sanger测序技术虽已成熟,但其速度和成本的限制满足不了大规模测序的要求。第二代高通量测序技术结合了乳胶微粒和皮升级反应的454焦磷酸测序法,作为一种高通量测序技术,具有分析结果准确、高速、高灵敏度和高自动化的特点。对454测序法的技术原理和操作步骤进行了介绍,对近年来运用该方法在环境微生物生态研究领域的进展进行了综述。     

Kinetic Modeling for Simultaneous Organic Carbon Oxidation,Nitrification, and Denitrification of Abattoir Wastewater in Sequencing Batch Reactor

A laboratory-scale study was conducted in a 20.0-L sequencing batch reactor (SBR) to explore the feasibility of simultaneous removal of organic carbon and nitrogen from abattoir wastewater. The reactor was operated under three different combinations of aerobic-anoxic sequence, viz., (4+4), (5+3), and (5+4) h of total react period, with influent soluble chemical oxygen demand (SCOD) and ammonia nitrogen (NH₄⁺-N) level of 2200 ± 50 and 125 ± 5 mg L^?1, respectively. In (5+4) h cycle, a maximum 90.27% of ammonia reduction corresponding to initial NH₄⁺-N value of 122.25 mg L^?1 and 91.36% of organic carbon removal corresponding to initial SCOD value of 2215.25 mg L^?1 have been achieved, respectively. The biokinetic parameters such as yield coefficient (Y), endogenous decay constant (k_d), and half-velocity constant (K_s) were also determined to improve the design and operation of package effluent treatment plants comprising SBR units. The specific denitrification rate (q_DN) during anoxic condition was estimated as 6.135 mg N/g mixed liquor volatile suspended solid (MLVSS)·h on 4-h average contact period. The value of Y, k_d and K_s for carbon oxidation and nitrification were found to be in the range of 0.6225–0.6952 mg VSS/mg SCOD, 0.0481–0.0588 day^?1, and 306.56–320.51 mg L^?1, and 0.2461–0.2541 mg VSS/mg NH₄⁺-N, 0.0324–0.0565 day^?1, and 38.28–50.08 mg L^?1, respectively, for different combinations of react periods.     

Autohydrogenotrophic Denitrifying Microbial Community in a Glass Beads Biofilm Reactor

A glass bead biofilm reactor was operated using H₂ as an electron donor to remove nitrate at 150 mg NO₃–N l⁻¹ to below detection level. The microbial community in the glass beads biofilm reactor was investigated by using denaturing gradient gel electrophoresis (DGGE) and phylogenetic analysis. In DGGE analysis of the biofilm, five bands were dominant and indicated the presence of eight β-proteobacteria, one γ-proteobacteria and twelve clostridia. An unculturable Hydrogenophaga sp., which is a new genus of hydrogen-oxidizing bacterium was dominant in microbial community of the biofilm reactor.     

Termite Hindguts and the Ecology of Microbial Communities in the Sequencing Age

Advances in high‐throughput nucleic acid sequencing have improved our understanding of microbial communities in a number of ways. Deeper sequence coverage provides the means to assess diversity at the resolution necessary to recover ecological and biogeographic patterns, and at the same time single‐cell genomics provides detailed information about the interactions between members of a microbial community. Given the vastness and complexity of microbial ecosystems, such analyses remain challenging for most environments, so greater insight can also be drawn from analysing less dynamic ecosystems. Here, we outline the advantages of one such environment, the wood‐digesting hindgut communities of termites and cockroaches, and how it is a model to examine and compare both protist and bacterial communities. Beyond the analysis of diversity, our understanding of protist community ecology will depend on using statistically sound sampling regimes at biologically relevant scales, transitioning from discovery‐based to experimental ecology, incorporating single‐cell microbiology and other data sources, and continued development of analytical tools.     

Microbial Community Profiles in Wastewaters from Onsite Wastewater Treatment Systems Technology

The aim of the study was to determine the potential of community-level physiological profiles (CLPPs) methodology as an assay for characterization of the metabolic diversity of wastewater samples and to link the metabolic diversity patterns to efficiency of select onsite biological wastewater facilities. Metabolic fingerprints obtained from the selected samples were used to understand functional diversity implied by the carbon substrate shifts. Three different biological facilities of onsite wastewater treatment were evaluated: fixed bed reactor (technology A), trickling filter/biofilter system (technology B), and aerated filter system (the fluidized bed reactor, technology C). High similarities of the microbial community functional structures were found among the samples from the three onsite wastewater treatment plants (WWTPs), as shown by the diversity indices. Principal components analysis (PCA) showed that the diversity and CLPPs of microbial communities depended on the working efficiency of the wastewater treatment technologies. This study provided an overall picture of microbial community functional structures of investigated samples in WWTPs and discerned the linkages between microbial communities and technologies of onsite WWTPs used. The results obtained confirmed that metabolic profiles could be used to monitor treatment processes as valuable biological indicators of onsite wastewater treatment technologies efficiency. This is the first step toward understanding relations of technology types with microbial community patterns in raw and treated wastewaters.     

Effects of Cycle Time on Properties of Aerobic Granules in Sequencing Batch Airlift Reactors

Summary The granulation and properties of aerobic sludge were studied in two sequencing batch airlift reactors (SBARs). The synthetic wastewater in the two reactors had initially different levels of COD (400 mg l⁻¹ in R1 and 1600 mg l⁻¹ in R2). A hydraulic cycle time of 3 and 12 h was conducted in the reactors R1 and R2, respectively and the process of granulation was observed by optical microscopy. It was found that the course of granulation at a cycle time of 3 h in R1 was shorter than that at cycle time of 12 h in R2 and the properties of aerobic granules were distinct in the reactors due to the different hydraulic cycle time. Under a cycle time of 3 h, granule diameter was around 1.0–2.0 mm, VSS ratio was 92.08% with stronger granule strength; under a cycle time of 12 h, granule diameter was around 0.5–1.0 mm, VSS ratio was 83.92% with weaker granule strength. In addition, the morphology of microorganisms in granules was obviously dissimilar when the hydraulic cycle time was different. It was concluded that the hydraulic cycle time plays a crucial role in the granulation and properties of aerobic granules. It is expected that the experimental findings will provide useful information on factors affecting aerobic granulation.     

造纸废水活性污泥的微生物群落结构及多样性分析

为探究造纸废水活性污泥中微生物群落结构多样性以及对造纸废水处理效果的影响，利用Illumina MiSeq 高通量测序方法，分析在处理造纸废水过程中，同一运行阶段两个并联氧化沟内活性污泥的微生物群落与多样性组成。结果表明，系统中处理造纸废水的活性污泥在同一废水条件下微生物群落结构总体稳定，优势细菌为绿弯菌门(Chloroflexi)、拟杆菌门（Bacteroidota）、变形菌门（Proteobacteria）、Myxococcota、放线菌门(Actinobacteria）、厚壁菌门(Firmicutes）等。最重要的优势细菌类群为Chloroflexi，相对丰度占比为47.67%~48.22%，远远高于其他废水中Chloroflexi的占比，其中厌氧绳菌纲（Anaerolineae）是其主要成员，占比84.39%~88.34%，可针对性地去除造纸废水中的污染物。造纸废水活性污泥样品中存在大量特殊功能菌群，其在废水中污染物尤其是木质素的去除中发挥着重要作用。     

High-Throughput Sequencing and Metagenomics: Moving Forward in the Culture-Independent Analysis of Food Microbial Ecology

Following recent trends in environmental microbiology, food microbiology has benefited from the advances in molecular biology and adopted novel strategies to detect, identify, and monitor microbes in food. An in-depth study of the microbial diversity in food can now be achieved by using high-throughput sequencing (HTS) approaches after direct nucleic acid extraction from the sample to be studied. In this review, the workflow of applying culture-independent HTS to food matrices is described. The current scenario and future perspectives of HTS uses to study food microbiota are presented, and the decision-making process leading to the best choice of working conditions to fulfill the specific needs of food research is described.