Chlorine-Susceptible and Chlorine-Resistant Type 021N Bacteria Occurring in Bulking Activated Sludges

Two filamentous bacteria causing bulking in two activated sludges were examined. Investigations using morphological features, staining techniques, and fluorescent in situ hybridization identified both filaments as type 021N. However, an examination of the effect of chlorine on the sludges revealed a chlorine-susceptible type 021N in one sludge and a chlorine-resistant type 021N in the other.     

Phylogenetic analysis of and oligonucleotide probe development for eikelboom type 021N filamentous bacteria isolated from bulking activated sludge

Fifteen filamentous strains, morphologically classified as Eikelboom type 021N bacteria, were isolated from bulking activated sludges. Based on comparative 16S ribosomal DNA (rDNA) sequence analysis, all strains form a monophyletic cluster together with all recognized Thiothrix species (88.3 to 98.7% 16S rDNA sequence similarity) within the gamma-subclass of Proteobacteria. The investigated Eikelboom type 021N isolates were subdivided into three distinct groups (I to III) demonstrating a previously unrecognized genetic diversity hidden behind the uniform morphology of the filaments. For in situ detection of these bacteria, 16S rRNA-targeted oligonucleotide probes specific for the entire Eikelboom type 021N-Thiothrix cluster and the Eikelboom type 021N groups I, II, and III, respectively, were designed, evaluated, and successfully applied in activated sludge.     

Quantification of an Eikelboom type 021N bulking event with fluorescence in situ hybridization and real-time PCR

Primers targeting 16S rRNA genes were designed to detect and quantify Eikelboom type 021N organisms by real-time PCR. Eikelboom type 021N filamentous bulking was induced in a laboratory-scale sequencing batch reactor and the evolution of Eikelboom type 021N 16S rRNA and 16S rRNA genes was monitored. A significant correlation was found between the sludge volume index and the amount of these filamentous organisms present in the sludge (r ²=94.6%, n=10, P<0.01), as measured by real-time PCR. The amount of Eikelboom type 021N 16S rRNA genes increased by a factor of 21 during the experiment, while the 16S rRNA increased by a factor of 33. Moreover, Eikelboom type 021N 16S rRNA increased with increased feeding frequency. It was observed that the RNA:DNA ratio peaked before the sludge volume index increased. In parallel, a fluorescence in situ hybridization study indicated a factor of four increase in the length of Eikelboom type 021N filaments, due to a factor of two increase in both length and number of Eikelboom type 021N filaments. Further, an increase in the fraction of filaments extending outside the activated sludge flocs was observed (19–55%). Monitoring of 16S rRNA genes and 16S rRNA of Eikelboom type 021N was shown to be valuable in evaluating activated sludge settling characteristics; and measuring RNA:DNA ratios may be used as an early warning tool for sludge bulking.     

Ultrastructure of Thiothrix spp. and "Type 021N" Bacteria

The ultrastructural features of two groups of filamentous sulfur bacteria, Thiothrix spp. and an unnamed organism designated "type 021N," were examined by transmission electron microscopy. Negative staining of whole cells and filaments with uranyl acetate revealed the presence of tufts of fimbriae located at the ends of individual gonidia of Thiothrix sp. strain A1 and "type 021N" strain N7. Holdfast material present at the center of mature rosettes was observed in thin sections stained with ruthenium red. A clearly defined sheath enveloped the trichomes of two of three Thiothrix strains but was absent from "type 021N" filaments. The outer cell wall appeared more complex in "type 021N" strains than in Thiothrix isolates. Bulbs or clusters of irregularly shaped cells, often present in filaments of "type 021N" bacteria, appeared to result from crosswalls which formed at angles oblique to the filament axis. The multicellular nature of these sulfur bacteria was apparent in that only the cytoplasmic membrane and peptidoglycan layer of the cell wall were involved in the septation process. Sulfur inclusions which developed in the presence of sodium thiosulfate were enclosed by a single-layered envelope and located within invaginations of the cytoplasmic membrane.     

Molecular phylogeny and taxonomy of colorless, filamentous sulfur bacteria of the genus Thiothrix

Comprehensive investigation combining molecular genetic techniques and comparative studies of morphological and physiological properties made it possible to resolve the disputed issue of the taxonomic status of the groups ??T. nivea?? and ??Eikelboom type 021N?? of the genus Thiothrix. The phylogenetic trees constructed on the basis of 16S rRNA and gyrB gene sequences demonstrated that members of the genus Thiothrix formed a cluster within the order Thiotrichales. According to the ??ribosomal?? tree, the cluster of the genus Thiothrix was divided into two main groups, I and II, corresponding to the groups ??T. nivea?? and ??Eikelboom type 021N??. The levels of similarity between the 16S rRNA gene sequences of Thiothrix species reached 88.9?C100%. On the contrary, in the ??gyrase?? tree, these species were not divided into ??T. nivea?? and ??Eikelboom type 021N?? groups. The levels of similarity between the amino acid sequences of the gyrB gene fragments of Thiothrix species varied from 74.5 to 99.2%. Importantly, members of the groups ??T. nivea?? and ??Eikelboom type 021N?? formed very similar 16S rRNA secondary structures in the variable region V3, where a 30-nucleotide deletion characteristic of all Thiothrix species was detected. Phenotypic analysis of the studied bacteria revealed some morphological and physiological properties shared by the groups ??T. nivea?? and ??Eikelboom type 021N??. The data obtained indicate that members of the groups ??T. nivea?? and ??Eikelboom type 021N?? are phenotypically and genetically heterogeneous species within the single monophyletic genus Thiothrix..     

Ultrastructure of Thiothrix spp. and “Type 021N” Bacteria

The ultrastructural features of two groups of filamentous sulfur bacteria, Thiothrix spp. and an unnamed organism designated “type 021N,” were examined by transmission electron microscopy. Negative staining of whole cells and filaments with uranyl acetate revealed the presence of tufts of fimbriae located at the ends of individual gonidia of Thiothrix sp. strain A1 and “type 021N” strain N7. Holdfast material present at the center of mature rosettes was observed in thin sections stained with ruthenium red. A clearly defined sheath enveloped the trichomes of two of three Thiothrix strains but was absent from “type 021N” filaments. The outer cell wall appeared more complex in “type 021N” strains than in Thiothrix isolates. Bulbs or clusters of irregularly shaped cells, often present in filaments of “type 021N” bacteria, appeared to result from crosswalls which formed at angles oblique to the filament axis. The multicellular nature of these sulfur bacteria was apparent in that only the cytoplasmic membrane and peptidoglycan layer of the cell wall were involved in the septation process. Sulfur inclusions which developed in the presence of sodium thiosulfate were enclosed by a single-layered envelope and located within invaginations of the cytoplasmic membrane.     

Indexes of assessing N availability in sewage sludges

Biological and chemical methods were used in an attempt to estimate N availability in sewage sludges. The two biological methods, i.e. maize plants grown in pots, and soil-sludge mixtures incubated at 2, 4, 6, 8, 12 and 16 weeks, and the four chemical methods, i.e. autoclave, 0.5 M KMnO₄, pepsin and 0.6 M HCl, were compared to determine N availability in twelve sewage sludges in a given soil. In the mineralization test, the aerobically treated sewage sludges gave higher mineralization rates than the anaerobically treated wastes. The simple correlation between available N, estimated from the plant N uptake during 6 weeks and N extracted by chemical methods showed that HCl and pepsin appeared to be the better single indexes. Prediction of availability of N in sewage sludges to plants in the growth chamber improved if N mineralized during the incubation period and extracted by several chemical methods were combined in a multiple regression analysis.     

The role of Trochilioides recta in elimination of bulking

Summary In the activated sludge treatment plant of Onoda City, bulking has often been caused by filamentous bacteria, types 021 N and I-2. Bulking disappeared when Trochilioides recta increased in the mixed liquor of the aeration tank. T.-recta was observed to ingest types 021 N or I-2. It may therefore be anticipated that this type of bulking could be controlled by T. recta.     

A quantitative method for measuring the mass concentration of the filamentous bacterium Type 021N in activated sludge using fluorescence in situ hybridization

AIMS: This study aimed to develop a quantitative method for measuring mass concentrations of Type 021N, a bacterium causing bulking in activated sludge. METHODS AND RESULTS: Fluorescence in situ hybridization was used to determine the relationship between the concentration ratio of the mass of the bacterium Type 021N to mass of activated sludge, and the proportion of fluorescence area imparted by probe G123T specific for Type 021N to that obtained with probe EUB338 for bacteria. A linear relationship existed between the cube root of the mass concentration ratio and square root of this area proportion. CONCLUSIONS: A standard curve was obtained for quantifying Type 021N in activated sludge. SIGNIFICANCE AND IMPACT OF THE STUDY: This method may allow the determination of growth rate constant of filamentous bacteria in activated sludge, information that will help in understanding their ecology.     

Effect of sludge type on poliovirus association with and recovery from sludge solids

Sludge type was found to affect the degree of association between seeded poliovirus type 1 (LSc) and sludge solids. The mean percent of solids-associated viruses for activated sludge mixed liquors, anaerobically digested sludges, and aerobically digested percent of solids-associated viruses for activated sludge mixed liquors, anaerobically digested sludges, and aerobically digested sludges was 57.2, 70.4, and 94.7, respectively. The degree of association between poliovirus and sludge solids was significantly greater for aerobically digested sludges than for the other two sludge types. Sludge solids associated viruses were eluted using 0.05 M glycine buffer, pH 10.5-11.0, and subsequently concentrated by organic flocculation. The effectiveness of the glycine method in the recovery of solids-associated viruses was also found to be affected by sludge type. Significantly lower mean poliovirus recovery was found for aerobically digested sludges (14.5%) than for mixed liquors or anaerobically digested sludges (72.3 and 60.2%, respectively). The eluent used in the method was not as effective in dissociating the virus from aerobic sludge solids as it was for the other two sludge types. All other virus adsorption-elution steps of the method (i.e., virus concentration steps) were equally effective in poliovirus recovery for all three sludge types. It is suggested that future methods developed for the recovery of viruses from sludges be evaluated for the various sludge types likely to be tested.     

Isolation and characterization of filamentous bacteria present in bulking activated sludge

Summary Several types of filamentous microorganisms were observed and identified in samples of poorly settling (bulking) activated sludge. The major types encountered and the frequency (percentage) of appearance in the total of all treatment plants sampled were: Eikelboom type 0041 (60), type 1701 (45), Haliscomenobacter hydrossis (35), type 021N (30), Thiothrix spp. (20), and Sphaerotilus natans (20). Isolation techniques and culture media were developed and used to recover 42 axenic strains of filamentous bacteria from sludge samples collected. The isolates were identified as strains of Thiothrix, Beggiatoa, S. natans, and Eikelboom types 021N, 1701, 0041, and 0803. Nutritional and differential characterization of the bacteria was important to the differentiation of groups which could not be easily distinguished on the basis of morphology. Although certain treatment plant operating parameters (organic loading) seemed associated with the presence of specific filamentous organism types, possible interaction among factors precluded definite establishment of a cause and effect relationship for most of the treatment plant characteristics and organisms observed.     

Filamentous sulfur bacteria of activated sludge: characterization of Thiothrix, Beggiatoa, and Eikelboom type 021N strains

Seventeen strains of filamentous sulfur bacteria were isolated in axenic culture from activated sludge mixed liquor samples and sulfide-gradient enrichment cultures. Isolation procedures involved plating a concentrated inoculum of washed filaments onto media containing sulfide or thiosulfate. The isolates were identified as Thiothrix spp., Beggiatoa spp., and an organism of uncertain taxonomic status, designated type 021N. All bacteria were gram negative, reduced nitrate, and formed long, multicellular trichomes with internal reserves of sulfur, volutin, and sudanophilic material. Thiothrix spp. formed rosettes and gonidia, and four of six strains were ensheathed. Type 021N organisms utilized glucose, lacked a sheath, and differed from Thiothrix spp. in several aspects of cellular and cultural morphology. Beggiatoa spp. lacked catalase and oxidase, and filaments were motile. Biochemical and physiological characterization of the isolates revealed important distinguishing features between the three groups of bacteria. Strain differences were most evident among the Thiothrix cultures. A comparison of the filamentous sulfur bacteria with freshwater strains of Leucothrix was made also.     

Filamentous sulfur bacteria of activated sludge: characterization of Thiothrix, Beggiatoa, and Eikelboom type 021N strains.

Seventeen strains of filamentous sulfur bacteria were isolated in axenic culture from activated sludge mixed liquor samples and sulfide-gradient enrichment cultures. Isolation procedures involved plating a concentrated inoculum of washed filaments onto media containing sulfide or thiosulfate. The isolates were identified as Thiothrix spp., Beggiatoa spp., and an organism of uncertain taxonomic status, designated type 021N. All bacteria were gram negative, reduced nitrate, and formed long, multicellular trichomes with internal reserves of sulfur, volutin, and sudanophilic material. Thiothrix spp. formed rosettes and gonidia, and four of six strains were ensheathed. Type 021N organisms utilized glucose, lacked a sheath, and differed from Thiothrix spp. in several aspects of cellular and cultural morphology. Beggiatoa spp. lacked catalase and oxidase, and filaments were motile. Biochemical and physiological characterization of the isolates revealed important distinguishing features between the three groups of bacteria. Strain differences were most evident among the Thiothrix cultures. A comparison of the filamentous sulfur bacteria with freshwater strains of Leucothrix was made also.     

Characterization of filamentous microorganisms in rotating biological contactor biofilms of wastewater treatment plants

Filamentous microorganisms populations present in biofilms from three wastewater treatment plants with a rotating biological contactor system have been studied. Results showed that filaments are an integral component of the biofilms, and that they were made up by: Beggiatoa ssp., Haliscomenobacter hydrossis, Sphaerotilus natans, Thiothrix ssp., and the 021N, 0803, 0914, 0961, 1701, 1851, and 1863 Eikelboom's types. Beggiatoa ssp. was the most frequent microorganism followed by S. natans and type 021N. Microthrix parvicella, Gordona amarae (=Nocardia amarae), Nostocoida limicola (I, II, III), and 0041, 0092, 0211, 0411, 0581, 0675, 1702, and 1852 Eikelboom's types appear infrequently.     

中国城市污泥有机质及养分含量与土地利用

土地利用是城市污泥的重要处理处置方法,其有机质和养分含量是土地利用中很受关注的问题。关于中国的城市污泥虽然已有不少研究报道,但不同研究结果之间结论并不一致,且缺乏系统的总结。通过系统搜集了20世纪80年代以来全国城市污泥相关的文献资料,重点评述中国城市污泥的有机质含量及其土地利用问题。通过29个城市污泥组成的统计分析发现,中国城市污泥(不包括工业污泥)的有机质平均含量达到384g／kg,全氮、全磷和全钾分别为27、14．3和7g／kg;有机质、全氮、全磷比纯猪粪分别高出1／3～2／3,但全钾比纯猪粪低1／3。中国城市污泥的有机质含量呈逐年增加的趋势,但氮、磷含量变化规律不明显。研究证明,城市污泥在粮食作物、蔬菜、经济作物及园林绿地上使用,具有明显的肥效和改良土壤的效果。近年来,城市污泥中重金属含量越来越低,这有利用促进城市污泥的土地利用,但中国在有机污染物和病原体的危害方面研究相对较少,对城市污泥土地利用的潜在风险仍缺乏长期的定位试验研究。     

Poliovirus retention in soil columns after application of chemical- and polyelectrolyte-conditioned dewatered sludges

The transport of poliovirus type 1 (strain LSc) was studied in Red Bay sandy loam columns that were treated with chemical- or polyelectrolyte-conditioned dewatered sludges and then leached with natural rainwater under saturated flow conditions. Poliovirus was concentrated in the alum and ferric chloride sludges that were produced following the flocculation of virus-seeded raw sewage. Virtually complete inactivation of the virus was observed following the flocculation of raw sewage or the stabilization of alum and ferric chloride sludges with lime at pH 11.5. Poliovirus was also concentrated in polyelectrolyte-conditioned dewatered sludge that was produced from virus-seeded, anaerobically digested sludge. Despite the saturated flow conditions for a sustained period, no viruses were detected in the leachates of the soil columns that were treated with these chemical and chemically treated sludges. Since the viruses were mostly associated with the solids in these sludge samples, it is believed that they were immobilized along with the sludge solids in the top portion of the soil columns.     

Poliovirus retention in soil columns after application of chemical- and polyelectrolyte-conditioned dewatered sludges.

The transport of poliovirus type 1 (strain LSc) was studied in Red Bay sandy loam columns that were treated with chemical- or polyelectrolyte-conditioned dewatered sludges and then leached with natural rainwater under saturated flow conditions. Poliovirus was concentrated in the alum and ferric chloride sludges that were produced following the flocculation of virus-seeded raw sewage. Virtually complete inactivation of the virus was observed following the flocculation of raw sewage or the stabilization of alum and ferric chloride sludges with lime at pH 11.5. Poliovirus was also concentrated in polyelectrolyte-conditioned dewatered sludge that was produced from virus-seeded, anaerobically digested sludge. Despite the saturated flow conditions for a sustained period, no viruses were detected in the leachates of the soil columns that were treated with these chemical and chemically treated sludges. Since the viruses were mostly associated with the solids in these sludge samples, it is believed that they were immobilized along with the sludge solids in the top portion of the soil columns.     

Quinone profiling of bacterial communities in natural and synthetic sewage activated sludge for enhanced phosphate removal

Respiratory quinones were used as biomarkers to study bacterial community structures in activated sludge reactors used for enhanced biological phosphate removal (EBPR). We compared the quinone profiles of EBPR sludges and standard sludges, of natural sewage and synthetic sewage, and of plant scale and laboratory scale systems. Ubiquinone (Q) and menaquinone (MK) components were detected in all sludges tested at molar MK/Q ratios of 0.455 to 0.981. The differences in MK/Q ratios were much larger when we compared different wastewater sludges (i.e., raw sewage and synthetic sewage) than when we compared sludges from the EBPR and standard processes or plant scale and laboratory scale systems. In all sludges tested a Q with eight isoprene units (Q-8) was the most abundant quinone. In the MK fraction, either tetrahydrogenated MK-8 or MK-7 was the predominant type, and there was also a significant proportion of MK-6 to MK-8 in most cases. A numerical cluster analysis of the profiles showed that the sludges tested fell into two major clusters; one included all raw sewage sludges, and the other consisted of all synthetic sewage sludges, independent of the operational mode and scale of the reactors and the phosphate accumulation. These data suggested that Q-8-containing species belonging to the class Proteobacteria (i.e., species belonging to the beta subclass) were the major constituents of the bacterial populations in the EBPR sludge, as well as in standard activated sludge. Members of the class Actinobacteria (gram-positive bacteria with high DNA G+C contents) were the second most abundant group in both types of sludge. The bacterial community structures in activated sludge processes may be affected more by the nature of the influent wastewater than by the introduction of an anaerobic stage into the process or by the scale of the reactors.     

Application of three methods used for the evaluation of polycyclic aromatic hydrocarbons (PAHs) bioaccessibility for sewage sludge composting

Three chemical approaches were compared to assess the bioaccessibility of PAHs during four sewage sludge composting. In the present study solid phase extraction with Tenax-TA and two non-exhaustive/mild-solvent extraction techniques with n-butanol (BtOH) and hydroxypropyl[beta]cyclodextrin (HPCD) have been applied. The content of the PAHs sum in individual sludges ranged from 2.83 to 9.95 mg/kg. Mainly 4-ring PAHs predominated in all sludges. The content of the bioaccessible fraction of PAHs determined using the BtOH and HPCD methods was usually twice higher than with the use of Tenax-TA adsorbent. The share of the bioaccessible fraction depended on the method applied and sewage sludge type. In the case of the PAH sums it ranged from 28.1% to 54.5% (Tenax-TA), from 25.7% to 80.9% (BtOH) and from 60.2% to 83.5% (HPCD). As a result of composting, the share of the bioaccessible fraction decreased by about 50% in most of the sludges examined. The reliable prediction of individual PAHs losses was observed for many of the PAHs; however, for a number of them, this was confirmed in more than two of the sludges examined.     

TT型病毒基因组的克隆与进化分析

利用PCR方法从输血传播性病毒 (transfusiontransmittedvirus,TTV)阳性标本中获得不同长度且重叠覆盖TTV基因组的DNA片段。将PCR扩增片段克隆到pT Adv载体中 ,筛选获得阳性克隆。DNA序列测定结果表明所克隆的片段为TTV基因组序列。利用DNA片段中特有的限制性内切酶位点将TTV的DNA片段首尾相连 ,得到近全长的基因组克隆 ,命名为TTV0 2 1。对TTV0 2 1的核酸序列进行分析 ,TTV0 2 1长 3472nt,存在 2个阅读框架ORF1和ORF2 ,分别编码 785和 1 46个氨基酸。将TTV0 2 1与其它已知的TTV基因组全序列进行了同源性比较 ,并进行进化分析。结果表明 ,TTV0 2 1序列与TTV分离株CHN2、BDH1的遗传距离较近 ,而与其它分离株相对较远。