Occurrence of ammonia-oxidizing archaea in wastewater treatment plant bioreactors

We report molecular evidence that ammonia-oxidizing archaea (AOA) occur in activated sludge bioreactors used to remove ammonia from wastewater. Using PCR primers targeting archaeal ammonia monooxygenase subunit A (amoA) genes, we retrieved and compared 75 sequences from five wastewater treatment plants operating with low dissolved oxygen levels and long retention times. All of these sequences showed similarity to sequences previously found in soil and sediments, and they were distributed primarily in four major phylogenetic clusters. One of these clusters contained virtually identical amoA sequences obtained from all five activated sludge samples (from Oregon, Wisconsin, Pennsylvania, and New Jersey) and accounted for 67% of all the sequences, suggesting that this AOA phylotype may be widespread in nitrifying bioreactors.     

Occurrence of Ammonia-Oxidizing Archaea in Wastewater Treatment Plant Bioreactors

We report molecular evidence that ammonia-oxidizing archaea (AOA) occur in activated sludge bioreactors used to remove ammonia from wastewater. Using PCR primers targeting archaeal ammonia monooxygenase subunit A (amoA) genes, we retrieved and compared 75 sequences from five wastewater treatment plants operating with low dissolved oxygen levels and long retention times. All of these sequences showed similarity to sequences previously found in soil and sediments, and they were distributed primarily in four major phylogenetic clusters. One of these clusters contained virtually identical amoA sequences obtained from all five activated sludge samples (from Oregon, Wisconsin, Pennsylvania, and New Jersey) and accounted for 67% of all the sequences, suggesting that this AOA phylotype may be widespread in nitrifying bioreactors.     

Occurrence of ammonia-oxidizing Archaea in activated sludges of a laboratory scale reactor and two wastewater treatment plants

Aims:  Characterization of the ammonia-oxidizing archaea (AOA) community in activated sludge from a nitrogen removal bioreactor and wastewater treatment plants (WWTPs).
Methods and Results:  Three primer sets specific for ammonia mono-oxygenase α -subunit ( amoA ) were used to construct clone libraries for activated sludge sample from a nitrogen removal bioreactor. One primer set resulted in strong nonspecific PCR products. The other two clone libraries retrieved both shared and unique AOA amoA sequences. One primer set was chosen to study the AOA communities of activated sludge samples from Shatin and Stanley WWTPs. In total, 18 AOA amoA sequences were recovered and compared to the previous reported sequences. A phylogenetic analysis indicated that sequences found in this study fell into three clusters.
Conclusions:  Different primers resulted in varied AOA communities from the same sample. The AOA found from Hong Kong WWTPs were closely similar to those from sediment and soil, but distinct from those from activated sludge in other places. A comparison of clone libraries between Shatin WWTP and bioreactor indicated the AOA community significantly shifted only after 30-day enrichment.
Significance and Impact of the Study:  This study confirmed the occurrence of AOA in a laboratory scale nitrogen removal bioreactor and Hong Kong WWTPs treating saline or freshwater wastewater. AOA communities found in this study were significantly different from those found in other places. To retrieve diverse AOA communities from environmental samples, a combination of different primers for the amoA gene is needed.     

A newly designed degenerate PCR primer based on pmoA gene for detection of nitrite-dependent anaerobic methane-oxidizing bacteria from different ecological niches

A new pmoA gene-based PCR primer set was designed for detection of nitrite-dependent anaerobic oxidation of methane (n-damo) bacteria from four different ecosystems, namely rice paddy soil, freshwater reservoir, reed bed, and sludge from wastewater treatment plant. This primer set showed high specificity and efficiency in recovering n-damo bacteria from these diverse samples. The obtained sequences showed 88–94 and 90–96 % similarity to nucleotide and amino acid sequences, respectively, with the known NC10 phylum bacterium. According to the UniFrac principal coordinates analysis (PCoA), DNA sequences retrieved by the new PCR primer set in this study formed a separate group from the reported sequences, indicating higher diversity of n-damo in the environment. This newly designed PCR primer is capable of amplifying not only the currently known n-damo bacteria but also those that have not been reported, providing new information on the ecological diversity and distribution of this group of microorganisms in the ecosystem.     

Characterization and cDNA cloning of midgut carboxypeptidases from Trichoplusia ni

Carboxypeptidase A and carboxypeptidase B activities from the midgut of Trichoplusia ni larvae were characterized. In the T. ni larval midgut, the primary digestive carboxypeptidase activity was attributed to carboxypeptidase A, which was eight times more active than carboxypeptidase B. Both the midgut carboxypeptidase A and carboxypeptidase B exhibited maximal activities at pH 8.0-8.5 and were similarly susceptible to inhibition by potato carboxypeptidase inhibitor and phenanthroline. The midgut carboxypeptidase activities were analyzed in T. ni larvae fed on various diet sources and the results indicated that midgut carboxypeptidase activities per milligram of gut were similar regardless of the amount of dietary proteins or amino acids. However, midgut carboxypeptidase A activity was significantly higher in larvae exposed to soybean trypsin inhibitor and was significantly lower in larvae fed on broccoli foliage. From the T. ni larval midgut, five putative carboxypeptidase cDNAs were cloned, demonstrating that midgut carboxypeptidase activities are composed of multiple carboxypeptidase types. Sequence analysis indicated that the midgut carboxypeptidases were produced as secreted proenzymes which could be activated after removal of an N-terminal activation fragment by a trypsin. Two cloned cDNAs are predicted to code for carboxypeptidase A and one cDNA is predicted to code for a putative carboxypeptidase B. The other two cDNAs are highly similar to carboxypeptidase A and carboxypeptidase B in sequences, but their activity was not predictable.     

Characterization of a digestive carboxypeptidase from the insect pest corn earworm (Helicoverpa armigera) with novel specificity towards C-terminal glutamate residues.

Carboxypeptidases were purified from guts of larvae of corn earworm (Helicoverpa armigera), a lepidopteran crop pest, by affinity chromatography on immobilized potato carboxypeptidase inhibitor, and characterized by N-terminal sequencing. A larval gut cDNA library was screened using probes based on these protein sequences. cDNA HaCA42 encoded a carboxypeptidase with sequence similarity to enzymes of clan MC [Barrett, A. J., Rawlings, N. D. & Woessner, J. F. (1998) Handbook of Proteolytic Enzymes. Academic Press, London.], but with a novel predicted specificity towards C-terminal acidic residues. This carboxypeptidase was expressed as a recombinant proprotein in the yeast Pichia pastoris. The expressed protein could be activated by treatment with bovine trypsin; degradation of bound pro-region, rather than cleavage of pro-region from mature protein, was the rate-limiting step in activation. Activated HaCA42 carboxypeptidase hydrolysed a synthetic substrate for glutamate carboxypeptidases (FAEE, C-terminal Glu), but did not hydrolyse substrates for carboxypeptidase A or B (FAPP or FAAK, C-terminal Phe or Lys) or methotrexate, cleaved by clan MH glutamate carboxypeptidases. The enzyme was highly specific for C-terminal glutamate in peptide substrates, with slow hydrolysis of C-terminal aspartate also observed. Glutamate carboxypeptidase activity was present in larval gut extract from H. armigera. The HaCA42 protein is the first glutamate-specific metallocarboxypeptidase from clan MC to be identified and characterized. The genome of Drosophila melanogaster contains genes encoding enzymes with similar sequences and predicted specificity, and a cDNA encoding a similar enzyme has been isolated from gut tissue in tsetse fly. We suggest that digestive carboxypeptidases with sequence similarity to the classical mammalian enzymes, but with specificity towards C-terminal glutamate, are widely distributed in insects.     

A novel rat carboxypeptidase, CPA2: characterization, molecular cloning, and evolutionary implications on substrate specificity in the carboxypeptidase gene family

A new member of the carboxypeptidase gene family, carboxypeptidase A2 (CPA2), has been identified from the predicted amino acid sequence of a rat pancreatic cDNA clone. In vivo recombination and in situ hybridization techniques employing the CPA2 cDNA resulted in the isolation of two genomic clones spanning the 25-kilobase pair rat CPA2 gene. Evolutionary trees built from the amino acid sequences of the known pancreatic carboxypeptidases show that CPA2 and carboxypeptidase A1 (CPA1) are the products of genes which duplicated before the mammalian radiation, and that bovine CPA is of the A1 type. The substrate specificities of CPA1 and CPA2 isolated from rat pancreas are similar to bovine CPA in that carboxyl-terminal amino acids with aromatic or branched aliphatic side chains are preferred. However, the substrate preference of rat CPA1 is skewed toward smaller amino acids, while that of rat CPA2 is skewed toward bulkier amino acids as compared to bovine CPA. The differences in the substrate specificities of these three carboxypeptidases are compatible with the nature of the amino acid replacements in their binding pockets for the carboxylterminal amino acid of the substrate.     

Metagenomic analysis of DNA viruses in a wastewater treatment plant in tropical climate

Viruses have been detected in the different stages of wastewater treatment plants (WWTPs) at concentrations of 10(8) -10(10) ml(-1) of virus-like particles (VLPs), 10-1000 times higher than in natural aquatic environments, suggesting that WWTPs can be considered as an important reservoir and source of viruses. This study revealed novel diversity and function with the DNA viral communities in the influent, activated sludge, anaerobic digester, and effluent of a domestic WWTP using metagenomics. WWTP was a very specific environment, with less than 5% of the >?936?000 metagenomic sequences obtained (～70-119?Mbp per sample) similar to sequences present in other environmental viromes. Many viruses found in the WWTP were novel, resulting in only ?82% were shared. These data suggested that VLPs of most viral types could be present between 1 and 30 days in the process before they were discharged. Viruses in WWTP and the discharged ones can have potential impacts on the functioning of the wastewater treatment system and on the dynamics of microbial community in the surrounding aquatic environments respectively.     

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。     

Identification of Diverse Antimicrobial Resistance Determinants Carried on Bacterial,Plasmid, or Viral Metagenomes from an Activated Sludge Microbial Assemblage

Using both sequence- and function-based metagenomic approaches, multiple antibiotic resistance determinants were identified within metagenomic libraries constructed from DNA extracted from bacterial chromosomes, plasmids, or viruses within an activated sludge microbial assemblage. Metagenomic clones and a plasmid that in Escherichia coli expressed resistance to chloramphenicol, ampicillin, or kanamycin were isolated, with many cloned DNA sequences lacking any significant homology to known antibiotic resistance determinants.Activated sludge in wastewater treatment plants is an open system with a dynamic and phylogenetically diverse microbial community (2, 3, 6, 7, 10, 11). Since the activated sludge process promotes cellular interactions among diverse microorganisms, there is great potential for the lateral transfer of antibiotic resistance genes between microbes in activated sludge and in downstream environments. Several studies have previously identified antibiotic resistance determinants from wastewater communities that are carried on bacterial chromosomes (1, 4, 14) and plasmids (9, 12, 13), but to our knowledge, a simultaneous metagenomic survey of antibiotic resistance determinants from all three genetic reservoirs (i.e., chromosomes, plasmids, and viruses) has never been performed within the same environment. To achieve a more comprehensive assessment of antibiotic resistance genes in the activated sludge microbial community, this study used both function- and sequence-based metagenomic approaches to identify antibiotic resistance determinants carried on bacterial chromosomes, plasmids, or viruses within an activated sludge microbial assemblage.     

The isolation and partial characterization of precursor forms of ostrich carboxypeptidase

Ostrich carboxypeptidases A and B were recently purified and characterized. The aim of this study was to isolate and purify, and partially characterize in terms of molecular weight, pI, amino acid composition and N-terminal sequencing, the precursor forms of carboxypeptidases from the ostrich pancreas. Inhibition studies with soybean trypsin inhibitor and activation studies with three proteases (bovine trypsin, bovine chymotrypsin and porcine elastase) were performed on crude ostrich acetone powder and the carboxypeptidase A and B activities were determined. SDS-PAGE was carried out after every incubation to monitor the rate and degree of conversion of a M(r) 66K component to procarboxypeptidase and carboxypeptidase A and B. The precursor forms were purified by Toyopearl Super Q and Pharmacia Mono Q chromatography. All three proteases converted the M(r) 66K component to procarboxypeptidases and carboxypeptidases over a set time interval, with carboxypeptidase A and B activities being detected in the acetone powder. Chymotrypsin was the preferred protease since it exhibited a more controlled activation of the procarboxypeptidases. The amino acid composition of procarboxypeptidase A revealed 525 residues. The N-terminal sequence of procarboxypeptidase A showed considerable homology when compared with several other mammalian sequences. M(r) and pI values of 52K and 5.23 were obtained for procarboxypeptidase A, respectively. This study indicated that ostrich procarboxypeptidase A is closely related to other mammalian procarboxypeptidase A molecules in terms of physicochemical properties.     

Changes in microbial diversity in fed-batch reactor operation with wastewater containing nitroaromatic residues

This study targeted the diversity of bacteria isolated from activated biomass of an ETP that treated wastewater from the dye industry. Phylogenetic analysis of 5'-partial 16S rDNA sequences revealed that the Proteobacteria (alpha, beta, and gamma) and Firmicutes were the major phyla in microbial community. Five unidentified culturable bacteria were also isolated. The activated biomass changed when acclimatized to wastewater with nitroaromatic residues. It was observed that the diversity of genera was reduced, but the degradative capacity of sludge was enhanced.     

Census of the Viral Metagenome within an Activated Sludge Microbial Assemblage

The viral metagenome within an activated sludge microbial assemblage was sampled using culture-dependent and culture-independent methods and compared to the diversity of activated sludge bacterial taxa. A total of 70 unique cultured bacterial isolates, 24 cultured bacteriophages, 829 bacterial metagenomic clones of 16S rRNA genes, and 1,161 viral metagenomic clones were subjected to a phylogenetic analysis.Bacteriophages play an active role in the ecology of natural environments, influencing prokaryotic population dynamics (5, 15) and mediating lateral gene transfer between diverse bacterial species, for example. Activated sludge (AS) microbial assemblages in wastewater treatment plants have been shown to harbor great numbers of viruses with a wide range of genome sizes (7, 9, 10, 16). Historically, the focus of wastewater viral studies has been on specific host-virus interactions, the application of phages as tools in microbial source tracking, or the use of phages to improve the efficiency of the wastewater treatment process (e.g., foam and pathogen control) (2, 4, 8, 12, 17). Despite the interest in the wastewater viral community, a census of the activated sludge total viral community has not, to our knowledge, been investigated using both culture-based and metagenomic approaches.     

活性污泥中菌群多样性及其功能调控研究进展

活性污泥是污水处理厂生物处理工艺的功能主体,活性污泥中菌群的种类、数量及活性是提高污水处理能力与效果的重要基础。本文综述了活性污泥处理工艺中的主要功能细菌(絮凝菌、脱氮菌、除磷菌等)生物群落的多样性与生态特征,并对目前主流的菌群鉴定方式进行总结,最后从运行条件、定向驯化及生物强化3个方面对菌群调控进行论述,以期为活性污泥法污水处理工艺提供一些理论指导。     

含氟有机废水处理过程活性污泥微生物群落研究进展

随着有机氟化物在各领域的广泛应用,含氟有机废水处理面临巨大挑战。活性污泥作为有机废水处理的核心技术之一,微生物在其中发挥着极其重要的作用。本综述首先聚焦在活性污泥微生物群落多样性、组成、结构和功能及其与含氟废水类型、处理工艺和处理效率之间的关系,进而讨论了功能微生物降解/转化有机氟化物的途径和作用机制,最后展望了结合分离培养降解有机氟化物的关键微生物,以及微生物组学技术解析活性污泥微生物群落构建、互作、代谢等核心问题,以提高对含氟有机废水微生物降解机理的认识,优化含氟有机废水处理工艺。     

Plasmid metagenome reveals high levels of antibiotic resistance genes and mobile genetic elements in activated sludge

The overuse or misuse of antibiotics has accelerated antibiotic resistance, creating a major challenge for the public health in the world. Sewage treatment plants (STPs) are considered as important reservoirs for antibiotic resistance genes (ARGs) and activated sludge characterized with high microbial density and diversity facilitates ARG horizontal gene transfer (HGT) via mobile genetic elements (MGEs). However, little is known regarding the pool of ARGs and MGEs in sludge microbiome. In this study, the transposon aided capture (TRACA) system was employed to isolate novel plasmids from activated sludge of one STP in Hong Kong, China. We also used Illumina Hiseq 2000 high-throughput sequencing and metagenomics analysis to investigate the plasmid metagenome. Two novel plasmids were acquired from the sludge microbiome by using TRACA system and one novel plasmid was identified through metagenomics analysis. Our results revealed high levels of various ARGs as well as MGEs for HGT, including integrons, transposons and plasmids. The application of the TRACA system to isolate novel plasmids from the environmental metagenome, coupled with subsequent high-throughput sequencing and metagenomic analysis, highlighted the prevalence of ARGs and MGEs in microbial community of STPs.     

Isolation and molecular cloning of mast cell carboxypeptidase A. A novel member of the carboxypeptidase gene family

Mast cell carboxypeptidase A has been isolated from the secretory granules of mouse peritoneal connective tissue mast cells (CTMC) and from a mouse Kirsten sarcoma virus-immortalized mast cell line (KiSV-MC), and a cDNA that encodes this exopeptidase has been cloned from a KiSV-MC-derived cDNA library. KiSV-MC-derived mast cell carboxypeptidase A was purified with a potato-derived carboxypeptidase-inhibitor affinity column and was found by analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be a Mr 36,000 protein. Secretory granule proteins from KiSV-MC and from mouse peritoneal CTMC were then resolved by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transblotted to polyvinylidine difluoride membranes. Identical aminoterminal amino acid sequences were obtained for the prominent Mr 36,000 protein present in the granules of both cell types. Based on the amino-terminal sequence, an oligonucleotide probe was synthesized and used to isolate a 1,470-base pair cDNA that encodes this mouse exopeptidase. The deduced amino acid sequence revealed that, after cleavage of a 15-amino acid hydrophobic signal peptide and a 94-amino acid activation peptide from a 417-amino acid preproenzyme, the mature mast cell carboxypeptidase A protein core has a predicted Mr of 35,780 and a high positive charge [Lys + Arg) - (Asp + Glu) = 17) at neutral pH. Although critical zinc-binding amino acids (His67, Glu70, His195), substrate-binding amino acids (Arg69, Asn142, Arg143, Tyr197, Asp255, Phe278), and cysteine residues that participate in intrachain disulfide bonds (Cys64-Cys77, Cys136-Cys159) of pancreatic carboxypeptidases were also present in mast cell carboxypeptidase A, the overall amino acid sequence identities for mouse mast cell carboxypeptidase A relative to rat pancreatic carboxypeptidases A1, A2, and B were only 43, 41, and 53%, respectively. RNA and DNA blot analyses revealed that mouse peritoneal CTMC, KiSV-MC, and bone marrow-derived mast cells all express a prominent 1.5-kilobase mast cell carboxypeptidase A mRNA which is transcribed from a single gene. We conclude that mouse mast cell carboxypeptidase A is a prominent secretory granule enzyme of mast cells of the CTMC subclass and represents a novel addition to the carboxypeptidase gene family.     

分子检测技术对活性污泥中氨氧化细菌的比较研究

采用PCR扩增、随机克隆测序等技术,分析处理含高浓度氨氮的废水处理系统不同驯化时期的4个活性污泥样品,对样品中氨氧化细菌（AOB）的种类和氨单加氧酶（AMO）的活性进行分析比较,并在国内首次采用PCR变性梯度凝胶电泳（DGGE）相结合的技术对样品中总的细菌类群的差异进行研究。结果表明所检测到的氨氧化细菌优势菌群均属于变形细菌的β亚类,与Nitrosomonas sp.具有较高的相似性。活性污泥驯化成熟后,废水处理系统中AMO的活性有明显提高,活性污泥中的细菌类群更加集中,优势菌群相对稳定,系统对废水的处理效率也相应提高。结果表明采用分子检测技术有利于更全面地了解AOB的类群和功能,进而改善废水处理系统的处理效果。     

In situ detection of starch-hydrolyzing microorganisms in activated sludge

Polysaccharides constitute a significant part of the organic matter in domestic wastewater and their hydrolysis plays an important role in their transformation and nutrient removal in activated sludge wastewater treatment plants. However, there is no information available about the identity, ecophysiology, and abundance of starch-hydrolyzing organisms (SHOs) in these plants. In this study, fluorescence in situ enzyme staining with BODIPY fluorescein-labeled starch was applied and optimized to label SHOs expressing alpha-amylase in activated sludge plants. Fluorescence on the surface of bacteria expressing alpha-amylase activity was clearly visualized. In 11 full-scale nutrient-removing wastewater treatment plants examined, the morphotypes of the dominant SHOs were always cocci in clusters of tetrads, short rods in clusters, and some filamentous organisms. The SHOs were identified by combining in situ enzyme staining and FISH using a range of available oligonucleotide probes. All the SHOs observed were Actinobacteria, and most had the phenotype of polyphosphate-accumulating organisms closely related to the genus Tetrasphaera in the family Intrasporangiaceae. The SHOs were present in most of the wastewater treatment plants examined and comprised, in total, up to 11% of bacterial biovolume and thus formed an important part of the microbial communities.     

Genetic diversity of genogroup IV noroviruses in wastewater in Japan

Aims: To investigate the prevalence, seasonality and genetic diversity of genogroup IV noroviruses (GIV NoVs) in wastewater in Japan. Methods and Results: Untreated and treated wastewater samples were collected monthly for a year from a wastewater treatment plant in Japan. The concentrated wastewater samples were examined for the presence of GIV NoV genomes with seminested RT‐PCR assay targeting partial capsid gene. Among 12 untreated and 12 treated wastewater samples tested, GIV NoV genomes were detected in three (25%) untreated and two (17%) treated wastewater samples with a high positive ratio in winter season. Genetic analysis revealed that the GIV NoVs in the wastewater samples were genetically diverse and were classified into three different genetic clusters. Conclusions: Frequent detection of GIV NoVs in winter season, which is a common epidemic period of human NoVs in Japan, indicates that GIV NoVs exhibit temporal trends similar to GI and GII NoVs. Based on the partial capsid gene sequences, we identified several unique GIV NoV strains belonging to the novel genetic cluster, demonstrating that GIV NoVs are more genetically diverse than previously appreciated. Significance and Impact of the Study: Our findings provide novel evidence of considerable genetic diversity among the GIV NoV strains.