活性污泥驯化的微生物生态学原理

活性污泥是一种复杂的、具有生物多样性的微生态系统.生物多样性是活性污泥驯化的基础,驯化条件对微生物进行选择--适者生存增长,不适者被抑制或淘汰.此外,活性污泥微生物还能够通过表型适应和进化性适应主动地去适应驯化条件.活性污泥驯化的过程是微生物生态位在生态系统中重新分配和调整的过程,符合生态学"选择与适应"的原理.依据这种原理,原污泥、废水水质和处理工艺是影响活性污泥驯化的主要因素.     

活性污泥驯化的微生物生态学原理

活性污泥是一种复杂的、具有生物多样性的微生态系统。生物多样性是活性污泥驯化的基础, 驯化条件对微生物进行选择—— 适者生存增长, 不适者被抑制或淘汰。此外, 活性污泥微生物还能够通过表型适应和进化性适应主动地去适应驯化条件。活性污泥驯化的过程是微生物生态位在生态系统中重新分配和调整的过程, 符合生态学“选择与适应”的原理。依据这种原理, 原污泥、废水水质和处理工艺是影响活性污泥驯化的主要因素。     

Microbial Ecology of Activated Sludge: II. Bacteriophages, Bdellovibrio, Coliforms, and Other Organisms

A comparative estimation of the coliform population of raw sewage, activated sludge, and the effluent derived therefrom revealed that raw sewage had a preponderance of Escherichia coli (75%), as compared with 25 and 30%, respectively, in sludge and effluent. Nitrogen-free mannitol-sucrose enrichments of activated sludge resulted in the isolation of Azotobacter agilis, Aerobacter aerogenes, Corynebacterium laevaniformans, and an Achromabacter species. Sludge had a large population of C. laevaniformans and A. aerogenes but not of Azotobacter. The bacterial parasites, Bdellovibrio and bacteriophages, were not active during activated-sludge treatment. A 10-fold reduction in phage content occurred after 2 hr of aeration, but the Bdellovibrio population was unaffected.     

Aerobic Heterotrophic Bacterial Populations of Sewage and Activated Sludge: II. Method of Characterization of Activated Sludge Bacteria

The replica-plating technique and Lochhead's nutritional method were combined in exploratory experiments to test their feasibility as useful means for characterizing the aerobic heterotrophic flora of activated sludge and to minimize the burdensome process of isolation, purification, and testing of isolates. In the test run, the method was about 86% reliable at the 0.05 level of significance. About 40% of the total number of bacteria able to grow on an aqueous extract of activated sludge did not grow on media containing glucose, amino acids, growth factors, and inorganic salts. The requirement for activated sludge extract suggested the existence of a requirement for unidentified nutrients contained in the activated sludge extract.     

Molecular Detection,Isolation, and Physiological Characterization of Functionally Dominant Phenol-Degrading Bacteria in Activated Sludge

DNA was isolated from phenol-digesting activated sludge, and partial fragments of the 16S ribosomal DNA (rDNA) and the gene encoding the largest subunit of multicomponent phenol hydroxylase (LmPH) were amplified by PCR. An analysis of the amplified fragments by temperature gradient gel electrophoresis (TGGE) demonstrated that two major 16S rDNA bands (bands R2 and R3) and two major LmPH gene bands (bands P2 and P3) appeared after the activated sludge became acclimated to phenol. The nucleotide sequences of these major bands were determined. In parallel, bacteria were isolated from the activated sludge by direct plating or by plating after enrichment either in batch cultures or in a chemostat culture. The bacteria isolated were classified into 27 distinct groups by a repetitive extragenic palindromic sequence PCR analysis. The partial nucleotide sequences of 16S rDNAs and LmPH genes of members of these 27 groups were then determined. A comparison of these nucleotide sequences with the sequences of the major TGGE bands indicated that the major bacterial populations, R2 and R3, possessed major LmPH genes P2 and P3, respectively. The dominant populations could be isolated either by direct plating or by chemostat culture enrichment but not by batch culture enrichment. One of the dominant strains (R3) which contained a novel type of LmPH (P3), was closely related to Valivorax paradoxus, and the result of a kinetic analysis of its phenol-oxygenating activity suggested that this strain was the principal phenol digester in the activated sludge.Many scientists have used the rRNA approach (29, 30) to detect microbial populations and to describe the structures of microbial communities in various environments without isolating the component microorganisms. These studies have shown that most 16S ribosomal DNA (rDNA) sequences directly amplified from environmental samples are different from the sequences of comparable laboratory strains. Workers have concluded from such observations that many bacteria that are predominant in the natural environment have not been isolated in the laboratory yet and that the microbial diversity in the natural environment is much greater than the diversity of the bacteria that have been isolated (2, 7, 13, 25, 35, 36, 39, 40).Currently, one important aspect of microbial ecology studies is functional dissection of microbial communities based on structural information obtained by the approach mentioned above. An analysis of a population shift accompanied by a change in the function of a community yields information useful for identifying functionally dominant populations (2, 3, 42), although information concerning the function (activity) of each population can never be obtained by this kind of approach. Hence, workers have emphasized that pure-culture experiments are indispensable for detailed analysis of the functions of each population and that isolation of the functionally dominant populations in a microbial community is quite important.Phenol and its derivatives are some of the major hazardous compounds in industrial wastewater (1, 31, 43), and for this reason biodegradation of phenol has attracted keen attention (34, 46). However, since most studies of phenol biodegradation have been carried out under laboratory conditions with arbitrarily selected phenol-degrading bacteria, phenol biodegradation in the environment is not well understood yet. In the present study, to better understand phenol degradation in activated sludge, we isolated and characterized the phenol-degrading bacteria that were identified by the rRNA approach to be the dominant population in phenol-digesting activated sludge. Physiological and genetic differences between the dominant phenol-degrading bacteria isolated in this study and representative phenol-degrading bacteria characterized previously in several laboratories are discussed below.     

Coaggregation among Nonflocculating Bacteria Isolated from Activated Sludge

Thirty-two strains of nonflocculating bacteria isolated from sewage-activated sludge were tested by a spectrophotometric assay for their ability to coaggregate with one other in two-membered systems. Among these strains, eight showed significant (74 to 99%) coaggregation with Acinetobacter johnsonii S35 while only four strains coaggregated, to a lesser extent (43 to 65%), with Acinetobacter junii S33. The extent and pattern of coaggregation as well as the aggregate size showed good correlation with cellular characteristics of the coaggregating partners. These strains were identified by sequencing of full-length 16S rRNA genes. A. johnsonii S35 could coaggregate with strains of several genera, such as Oligotropha carboxidovorans, Microbacterium esteraromaticum, and Xanthomonas spp. The role of Acinetobacter isolates as bridging organisms in multigeneric coaggregates is indicated. This investigation revealed the role of much-neglected nonflocculating bacteria in floc formation in activated sludge.     

Combined Molecular and Conventional Analyses of Nitrifying Bacterium Diversity in Activated Sludge: Nitrosococcus mobilis and Nitrospira-Like Bacteria as Dominant Populations

The ammonia-oxidizing and nitrite-oxidizing bacterial populations occurring in the nitrifying activated sludge of an industrial wastewater treatment plant receiving sewage with high ammonia concentrations were studied by use of a polyphasic approach. In situ hybridization with a set of hierarchical 16S rRNA-targeted probes for ammonia-oxidizing bacteria revealed the dominance of Nitrosococcus mobilis-like bacteria. The phylogenetic affiliation suggested by fluorescent in situ hybridization (FISH) was confirmed by isolation of N. mobilis as the numerically dominant ammonia oxidizer and subsequent comparative 16S rRNA gene (rDNA) sequence and DNA-DNA hybridization analyses. For molecular fine-scale analysis of the ammonia-oxidizing population, a partial stretch of the gene encoding the active-site polypeptide of ammonia monooxygenase (amoA) was amplified from total DNA extracted from ammonia oxidizer isolates and from activated sludge. However, comparative sequence analysis of 13 amoA clone sequences from activated sludge demonstrated that these sequences were highly similar to each other and to the corresponding amoA gene fragments of Nitrosomonas europaea Nm50 and the N. mobilis isolate. The unexpected high sequence similarity between the amoA gene fragments of the N. mobilis isolate and N. europaea indicates a possible lateral gene transfer event. Although a Nitrobacter strain was isolated, members of the nitrite-oxidizing genus Nitrobacter were not detectable in the activated sludge by in situ hybridization. Therefore, we used the rRNA approach to investigate the abundance of other well-known nitrite-oxidizing bacterial genera. Three different methods were used for DNA extraction from the activated sludge. For each DNA preparation, almost full-length genes encoding small-subunit rRNA were separately amplified and used to generate three 16S rDNA libraries. By comparative sequence analysis, 2 of 60 randomly selected clones could be assigned to the nitrite-oxidizing bacteria of the genus Nitrospira. Based on these clone sequences, a specific 16S rRNA-targeted probe was developed. FISH of the activated sludge with this probe demonstrated that Nitrospira-like bacteria were present in significant numbers (9% of the total bacterial counts) and frequently occurred in coaggregated microcolonies with N. mobilis.     

Isolation and Characterization of Cellulolytic Bacteria from Activated Sludge

Cellulolytic aerobic bacteria were isolated from activated sludge systems. Of the media tested for enumeration, only filter paper media gave reliable counts. Five isolates were studied further for characterization. It was found that one strain (DK) belonged to the genus Cellulomonas. The other four strains expressed similarity to the genus Pseudomonas. The different characteristics that were studied, however, do not permit them to be identified with any recognized species. Based on certain characters we believe that they are alcaligenes-like pseudomonads.     

污水处理活性污泥微生物群落多样性研究

为研究污水处理活性污泥微生物多样性,提取了活性污泥宏基因组DNA,并采用细菌通用引物27F和1492R扩增了上海污泥厂活性污泥细菌16S rDNA片段,构建了细菌16S rDNA克隆文库,并对该文库中的微生物群落进行了分析。共获得200条高质量序列并建立系统发育树,结果显示活性污泥主要的细菌类群为变形菌门(Proteobacteria)(91.9%)、厚壁菌门(Firmicures)(4.6%)、拟杆菌门(Bacteroidetes)(2%)、绿弯菌门(Chloroflexi)(0.5%)、硝化螺菌门(Nitrospirae)(1%)。其中,明显的优势菌群为Alcaligenes feacalis(55%)、Pseudomonas aeruginosa(12.8%)和Stenotrophomonas(12.8%),优势菌的产酶能力在活性污泥中显示生态修复功能菌的作用。     

An Evaluation of Procedures for Enumerating Bacteria in Activated Sludge

S ummary : A procedure for counting viable heterotrophic bacteria in activated sludge was evolved from a study of the effects of modifications to procedures at the different stages of enumeration. Optimal counts were obtained with Casitone-glycerol-yeast extract agar (CGY) with incubation for 6 days at 22°. Homogenization of mixed liquor was conveniently performed, with minimal lethal effect on the bacteria, by treating samples, diluted 1/10 in sodium tripolyphosphate solution (5 mg/1), in a boiling tube immersed in the Kerry ultrasonic cleaning bath for 1 min. Counts were significantly affected by the pH value of diluent and CGY, but not by the homogenization method or by treating homogenized samples with enzymes or N -acetyl cysteine, or by adding colloidal peptizing agents to the diluent. Replicate colony counts showed variances greater than the mean, although precision increased with increasing number of colonies/dish; there was a direct relationship between colony counts and volume plated for up to c. 1000 colonies/dish. Counts on spread plates tended to be higher and more precise than on dilution frequency plates, although the 2 methods showed satisfactory correlation. Counts were not significantly affected by the method of sampling and preparing the initial dilution, and it was considered prudent to examine samples immediately after collection.     

Aerobic Heterotrophic Bacterial Populations of Sewage and Activated Sludge: I. Enumeration1

Agar plating media containing solely activated sludge extracts yielded, in general, higher viable counts of activated sludge bacteria than any other culture medium tested. Activated sludge extracts made from different treatment plants varied in efficacy in evoking maximal viable counts. Frequently, homologous plating, i.e., plating inocula of activated sludges on extracts made from the same activated sludges, tended to yield lower counts than the heterologous platings tried in this investigation. The counts obtained by homologous plating of activated sludge were not significantly lower and sometimes were even significantly higher than the counts obtained on standard Nutrient Agar, which had been found by previous workers to be a good medium for counting activated sludge bacteria. The higher counts obtained with activated sludge extracts set objectives for formulating reproducible or defined culture media for the enumeration of activated sludge bacteria.     

有机农药活性污泥中表面活性剂产生菌的筛选及鉴定

目的:筛选表面活性剂产生菌,用于降解有机农药,提高氧化塘处理效率.方法:以液体石蜡为惟一碳源进行选择性培养,通过测定发酵液的排油活性和表面张力,对有机农药厂的活性污泥进行产生物表面活性剂细菌筛选,并对筛得的细菌进行形态学、生理生化及 16SrDNA 试验和鉴定.结果:筛选出 3 株产生物表面活性剂的细菌,经鉴定均属于不动杆菌属.结论:证实了有机农药活性污泥中存在表面活性剂的产生菌,对有机农药降解存在助溶作用.     

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.     

Simultaneous Fluorescent Gram Staining and Activity Assessment of Activated Sludge Bacteria

Wastewater treatment is one of the most important commercial biotechnological processes, and yet the component bacterial populations and their associated metabolic activities are poorly understood. The novel fluorescent dye hexidium iodide allows assessment of Gram status by differential absorption through bacterial cell walls. Differentiation between gram-positive and gram-negative wastewater bacteria was achieved after flow cytometric analysis. This study shows that the relative proportions of gram-positive and gram-negative bacterial cells identified by traditional microscopy and hexidium iodide staining were not significantly different. Dual staining of cells for Gram status and activity proved effective in analyzing mixtures of cultured bacteria and wastewater populations. Levels of highly active organisms at two wastewater treatment plants, both gram positive and gram negative, ranged from 1.5% in activated sludge flocs to 16% in the activated sludge fluid. Gram-positive organisms comprised <5% of the total bacterial numbers but accounted for 19 and 55% of the highly active organisms within flocs at the two plants. Assessment of Gram status and activity within activated sludge samples over a 4-day period showed significant differences over time. This method provides a rapid, quantitative measure of Gram status linked with in situ activity within wastewater systems.     

Amyloid-Like Adhesins Produced by Floc-Forming and Filamentous Bacteria in Activated Sludge

Amyloid proteins (fimbriae or other microbial surface-associated structures) are expressed by many types of bacteria, not yet identified, in biofilms from various habitats, where they likely are of key importance to biofilm formation and biofilm properties. As these amyloids are potentially of great importance to the floc properties in activated sludge wastewater treatment plants (WWTP), the abundance of amyloid adhesins in activated sludge flocs from different WWTP and the identity of bacteria producing these were investigated. Amyloid adhesins were quantified using a combination of conformationally specific antibodies targeting amyloid fibrils, propidium iodide to target all fixed bacterial cells, confocal laser scanning microscopy, and digital image analysis. The biovolume fraction containing amyloid adhesins ranged from 10 to 40% in activated sludge from 10 different WWTP. The identity of bacteria producing amyloid adhesins was determined using fluorescence in situ hybridization with oligonucleotide probes in combination with antibodies or thioflavin T staining. Among the microcolony-forming bacteria, amyloids were primarily detected among Alpha- and Betaproteobacteria and Actinobacteria. A more detailed analysis revealed that many denitrifiers (from Thauera, Azoarcus, Zoogloea, and Aquaspirillum-related organisms) and Actinobacteria-related polyphosphate-accumulating organisms most likely produced amyloid adhesins, whereas nitrifiers did not. Many filamentous bacteria also expressed amyloid adhesins, including several Alphaproteobacteria (e.g., Meganema perideroedes), some Betaproteobacteria (e.g., Aquaspirillum-related filaments), Gammaproteobacteria (Thiothrix), Bacteroidetes, Chloroflexi (e.g., Eikelboom type 1851), and some foam-forming Actinobacteria (e.g., Gordonia amarae). The results show that amyloid adhesins were an abundant component of activated sludge extracellular polymeric substances and seem to have unexpected, divers functions.     

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。