Bacterial Competition in Activated Sludge: Theoretical Analysis of Varying Solids Retention Times on Diversity

A mechanistic model for activated sludge sewage treatment was developed to predict exploitative competition of six aerobic heterotrophic bacterial species competing for three essential resources. The central hypothesis of the model is that in a multispecies/limiting resource system the number of coexisting bacterial species, N, exceeds the number of limiting resources, K, available for them. The explanation for this is that for certain species combinations, the dynamics of the competition process generate oscillations in the abundances of species, and these oscillations allow the coexistence of greater number of species than the number of limiting resources (N > K). This result is a direct contradiction of an existing activated sludge steady state competition theory, the principle of competitive exclusion, which states that the competition process proceeds to equilibrium, allowing only N K species to coexist. The model was used to investigate the effect of varying solids retention times on the diversity of species using the conventional, completely mixed activated sludge configuration. The results of model simulations showed that for a certain range of solids retention times (2.28–5.66 days) the competition of six species for three essential resources produces oscillations within the structure of the bacterial community allowing for the sustained growth of more than three species on three resources.This revised version was published online in November 2004 with corrections to Volume 48.     

Diversity of Dominant Bacterial Taxa in Activated Sludge Promotes Functional Resistance following Toxic Shock Loading

Examining the relationship between biodiversity and functional stability (resistance and resilience) of activated sludge bacterial communities following disturbance is an important first step towards developing strategies for the design of robust biological wastewater treatment systems. This study investigates the relationship between functional resistance and biodiversity of dominant bacterial taxa by subjecting activated sludge samples, with different levels of biodiversity, to toxic shock loading with cupric sulfate (Cu[II]), 3,5-dichlorophenol (3,5-DCP), or 4-nitrophenol (4-NP). Respirometric batch experiments were performed to determine the functional resistance of activated sludge bacterial community to the three toxicants. Functional resistance was estimated as the 30 min IC₅₀ or the concentration of toxicant that results in a 50% reduction in oxygen utilization rate compared to a referential state represented by a control receiving no toxicant. Biodiversity of dominant bacterial taxa was assessed using polymerase chain reaction-terminal restriction fragment length polymorphism (PCR-T-RFLP) targeting the 16S ribosomal RNA (16S rRNA) gene. Statistical analysis of 30 min IC₅₀ values and PCR-T-RFLP data showed a significant positive correlation (P < 0.05) between functional resistance and microbial diversity for each of the three toxicants tested. To our knowledge, this is the first study showing a positive correlation between biodiversity of dominant bacterial taxa in activated sludge and functional resistance. In this system, activated sludge bacterial communities with higher biodiversity are functionally more resistant to disturbance caused by toxic shock loading.     

制浆造纸生物技术研究进展

制浆造纸工业是国民经济的主要支柱之一,但也是资源消耗和环境污染的大户。近年来,国外用于制浆造纸工业的生物技术研究异常活跃,除废水生物处理外,木聚糖酶助漂、脂肪酶控制树脂、木片真菌预处理和酶法废纸脱墨等工艺已经在生产中得到实际应用,生物制浆、漆酶漂白工艺也已进入中试阶段。结合以草浆为主的特点,我国的制浆造纸生物技术研究也已日趋活跃起来。     

Bio-prospecting Bacterial Diversity of Hot Springs in Northern Himalayan Region of India for Laccases

Bacterial diversity of hot springs of northern Himalayan region of India was studied and explored for laccases, the multicopper enzymes applicable in a large number of industries due to their ability to utilize a wide range of substrates. 220 operational taxonomic units (OTUs) out of 5551 sequence reads for bacterial diversity and 3 OTUs out of 19 sequence reads for Laccase like multicopper oxidases (LMCOs) diversity were generated. Bacteroidetes (74.28%) was the most abundant phylum including genus Paludibacter (66.96%), followed by phylum Proteobacteria (24.53%) including genera Chitinilyticum (7.55%) and Cellvibrio (6.14%). In case of laccase diversity, three LMCO sequences showed affiliation with proteobacteria and one with two domain laccase from uncultivable bacteroidetes. LMCO sequences belonged to H and N families.     

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.     

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。     

Pulp Nonfiction: Regionalized Dynamic Model of the U.S. Pulp and Paper Industry

This article presents a modeling framework that enhances our ability to analyze the implications of policy for future sustainability of industrial systems. The framework quantifies the relationship between physical input and waste flows, capital vintage, and investment behavior in the U.S. pulp and paper industry. A regional vintage model is developed that simultaneously incorporates investment decisions, vintage structure of the capital stock, and physical material and energy flows, in addition to paper demand. Each capital vintage is specified by size, output structure, and age-specific retirement rates, as well as fiber use and energy intensities. Both embodied and disembodied technological change are incorporated, as well as greenhouse gas emissions from fuel use, and decomposition and incineration of waste. Estimated equations are used to simulate industrial futures until 2020, from a system of nonlinear differential equations.
Our results demonstrate the economic and physical inter-dependence between material and energy flows and the central role energy prices have in decision-making. For instance, an increase in average energy prices, ceteris paribus , will on average discourage paper recycling, which has implications for greenhouse gas emissions as well as for changes in energy intensity. The analysis of the data reveals diminishing rates of energy self-generation, and the immense longevity of capital, which hampers rapid change in input and carbon intensity. This stresses the importance of investment-led strategies in facilitating faster capital turnover to enhance future sustainability of the system.     

Mechanisms Prevalent during Bioremediation of Wastewaters from the Pulp and Paper Industry

Bioremediation of wastewaters represents an important treatment methodology, especially when examined against the backdrop of ever-stricter legislation that is evolving in order to regulate effluent release into the environment. It has been reported that bioremediation specifically holds promise in solving environmental problems. Crucial questions surrounding the treatment of effluents include: efficiency of the process, economic feasibility, legal requirements, and the mechanisms involved in the remediation process. Of all these issues mentioned, the last requires special attention. This paper investigates these matters and focuses on techniques that are currently employed to determine the efficiency of bioremediation and mechanisms involved therein. The physiological significance of biosorption is also examined, as this subject has not been fully addressed in previous publications.     

Aerobic Heterotrophic Bacterial Populations of Sewage and Activated Sludge: IV. Adaptation of Activated Sludge to Utilization of Aromatic Compounds 1

An activated sludge from a sewage treatment plant and a laboratory activated sludge developed on an artificial waste were compared for their ability to utilize 11 aromatic compounds. There were several significant differences between them. The laboratory sludge contained higher numbers of organisms and metabolized the aromatics to a greater extent. Laboratory activated sludges acclimated to utilization of the aromatics differed from each other in population structure and the pattern of oxygen consumption with aromatic substrates. The oxidative patterns of uncontrolled mixed populations were unreliable for investigating metabolic pathways. Extracts of the various sludges elevated the plate counts of the sludges.     

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。     

Evolution of Bacterial Diversity during Enrichment of PCP-Degrading Activated Soils

The microbiota of completely mixed soil slurry was acclimated with pentachlorophenol (PCP) or with a wood preservative mixture (WPM) containing several pollutants such as PCP and petroleum hydrocarbons. The impact of these compounds on the bacterial diversity was studied by using molecular tools. PCR amplifications of the 16S ribosomal RNA gene sequences (rDNA) were carried out with total DNA extracted from soil slurry samples taken at different time points during the enrichment process of the PCP and WPM reactors. The composition of these PCR products, reflecting the bacterial diversity, was monitored by the single-strand-conformation polymorphism (SSCP) method. Our results showed that the complexity of the SSCP profiles in the PCP reactor decreased significantly during the enrichment process, whereas they remained complex in the WPM reactor. PCR-amplified 16 rDNA libraries were generated from each reactor. The SSCP method was used to rapidly screen several clones of these libraries to find specific single-strand DNA migration profiles. In the PCP-activated soil, 96% of examined clones had the same SSCP profile, and sequences of representative clones were related to the genusSphingomonas, suggesting that the enrichment with PCP resulted in a selection of little phylogenetic diversity. Four different SSCP profiles were observed with the 68 examined clones from the WPM reactor. Representative clones of these profiles were related to Methylocystaceae or Rhizobiaceae, to sulfur-oxidizing symbionts, to the genusAcinetobacter, and to the genusSphingomonas. We also cloned and sequenced PCR-amplified DNA related to thepcpB gene, coding for theSphingomonas PCP-4-monooxygenase and detected in both reactors after two weeks of enrichment. Of the 16 examined clones, deduced amino acid sequences of 13 clones were highly related to theSphingomonas sp. strain UG30pcpB. The three remainingpcpB clones were not closely related to the three knownSphingomonas pcpB.     

Abundance, Diversity, and Dynamics of Viruses on Microorganisms in Activated Sludge Processes

We examined the abundance of viruses on microorganisms in activated sludge and the dynamics of their community structure. Direct counting with epifluorescence microscopy and pulsed-field gel electrophoresis (PFGE) were applied to 20 samples from 14 full-scale wastewater treatment plants (wwtps) treating municipal, industrial, or animal wastewater. Furthermore, to observe the dynamics of viral community structure over time, a laboratory-scale sequencing batch reactor was operated for 58 days. The concentrations of virus particles in the wwtps, as quantified by epifluorescence microscopy, ranged from 4.2 × 10⁷ to 3.0 × 10⁹ mL⁻¹. PFGE, improved by the introduction of a higher concentration of Tris–EDTA buffer in the DNA extraction step, was successfully used to profile DNA viruses in the activated sludge. Most of the samples from different wwtps commonly had bands in the 40–70 kb range. In the monitoring of viral DNA size distribution in the laboratory-scale reactor, some bands were observed stably throughout the experimental period, some emerged during the operation, and others disappeared. Rapid emergence and disappearance of two intense bands within 6 days was observed. Our data suggest that viruses—especially those associated with microorganisms—are abundant and show dynamic behavior in activated sludge.     

厌氧污泥体系脱氢酶活性表征细菌数的研究

以硫化钠作还原剂,甲苯为提取溶剂,用氯化三苯基四氮唑(TTC)法测定污泥脱氢酶活性,探讨了脱氢酶活性与细菌数对数(lgABN)的相关性,并建立了脱氢酶活性表征细菌数的数学模型。结果表明:在lgABN为7.5～12范围内,脱氢酶表征的污泥活性与细菌数对数(lgABN)呈明显线性相关,从理论上分析了其惟一线性相关性;在细菌数的对数达到7.5～12的对数期、稳定期与衰亡期,脱氢酶活性换算成对应细菌数所绘制的曲线与细菌生长曲线有明显拟合;在有重金属离子存在的污泥体系中,脱氢酶活性可用来作为评价重金属离子的毒性指标;从而脱氢酶活性可取代细菌计数表征污泥活性。     

Localization and Identification of Populations of Phosphatase-Active Bacterial Cells Associated with Activated Sludge Flocs

Abstract The majority of phosphatase (PO₄ase) activity detected in fresh aerobic activated sludge from a municipal wastewater treatment plant was associated with suspended floc material. PO₄ase activity appeared to be localized in discrete bacteria-containing areas of the floc matrix based on the distribution of nucleic acid–stained cells and precipitated fluorescent crystals produced as a result of reaction of the enzyme(s) with the artificial substrate ELF™-PO₄. Of the total floc-associated bacterial cells that stained positive with the nucleic acid–binding fluorochrome acridine orange (AO), 8.8 ± 1.2% displayed PO₄ase activity based on the proximity of AO-stained cells to precipitated ELF crystals. Using a 16S rRNA oligonucleotide probe specific for the cytophaga–flavobacteria group, it was determined that 17–20% of the floc-associated bacteria that probed positive also displayed PO₄ase activity. Furthermore, 35–45% of the ELF fluorescence was associated with bacterial cells that probed positive for the cytophaga–flavobacteria group. The results suggest that the cytophaga–flavobacteria, as a group, is important in mediating the liberation of inorganic orthophosphate (P_i) from phosphomonoesters of detrital organic phosphate (organic-P) in the aerobic activated sludge process of wastewater treatment. Received: 17 March 1999; Accepted: 9 June 1999     

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.     

Use of 16S rRNA Gene Terminal Restriction Fragment Analysis To Assess the Impact of Solids Retention Time on the Bacterial Diversity of Activated Sludge

Terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes was used to investigate the reproducibility and stability in the bacterial community structure of laboratory-scale sequencing batch bioreactors (SBR) and to assess the impact of solids retention time (SRT) on bacterial diversity. Two experiments were performed. In each experiment two sets of replicate SBRs were operated for a periods of three times the SRT. One set was operated at an SRT of 2 days and another set was operated at an SRT of 8 days. Samples for T-RFLP analysis were collected from the two sets of replicate reactors. HhaI, MspI, and RsaI T-RFLP profiles were analyzed using cluster analysis and diversity statistics. Cluster analysis with Ward's method using Jaccard distance and Hellinger distance showed that the bacterial community structure in both sets of reactors from both experimental runs was dynamic and that replicate reactors were clustered together and evolved similarly from startup. Richness (S), evenness (E), the Shannon-Weaver index (H), and the reciprocal of Simpson's index (1/D) were calculated, and the values were compared between the two sets of reactors. Evenness values were higher for reactors operated at an SRT of 2 days. Statistically significant differences in diversity (H and D) between the two sets of reactors were tested using a randomization procedure, and the results showed that reactors from both experimental runs that were operated at an SRT of 2 days had higher diversity (H and D) at the 5% level. T-RFLP analysis with diversity indices proved to be a powerful tool to analyze changes in the bacterial community diversity in response to changes in the operational parameters of activated-sludge systems.