Removal of Escherichia coli in wastewater by activated sludge.

Removal of bacteria from wastewater treated with activated sludge was studied by the use of a streptomycin-resistant Escherichia coli strain. The removal appeared to be a biphasic process. A rapid sorption of bacteria to the sludge flocs took place in the first hour after seeding mixed liquor with E. coli. Thereafter, slower elimination of E. coli was observed. The latter process was due to predation on E. coli by ciliated protozoa. This was shown by: (i) appearance of fluorescent food vacuoles of ciliates when fluorescent E. coli cells were added to mixed liquor; (ii) inhibition of predation either in the presence of cycloheximide or under anaerobic conditions; and (iii) absence of predation in bulking and washed sludge.     

Removal of Escherichia coli in wastewater by activated sludge.

Removal of bacteria from wastewater treated with activated sludge was studied by the use of a streptomycin-resistant Escherichia coli strain. The removal appeared to be a biphasic process. A rapid sorption of bacteria to the sludge flocs took place in the first hour after seeding mixed liquor with E. coli. Thereafter, slower elimination of E. coli was observed. The latter process was due to predation on E. coli by ciliated protozoa. This was shown by: (i) appearance of fluorescent food vacuoles of ciliates when fluorescent E. coli cells were added to mixed liquor; (ii) inhibition of predation either in the presence of cycloheximide or under anaerobic conditions; and (iii) absence of predation in bulking and washed sludge.     

Effect of oxygen concentration on nitrification and denitrification in single activated sludge flocs

Simultaneous nitrification and denitrification (SND) was investigated in the single aeration tank of a municipal wastewater treatment plant. Microelectrode measurements and batch experiments were performed to test for the presence of SND. Microelectrodes recorded the presence of O(2) concentration gradients in individual activated sludge flocs. When the O(2) concentration in the bulk liquid was <45 microM, anoxic zones were detected within flocs with a larger diameter (approximately 3000 microm). The O(2) penetration depth in the floc was found to be dependent on the O(2) concentration in the bulk liquid. Nitrification was restricted to the oxic zones, whereas denitrification occurred mainly in the anoxic zones. The nitrification rate of the activated sludge increased with increasing O(2) concentration in the bulk liquid, up to 40 microM, and remained constant thereafter. SND was observed in the aerated activated sludge when O(2) concentration was in the range of 10 to 35 microM.     

Impact of protozoan grazing on nitrification and the ammonia- and nitrite-oxidizing bacterial communities in activated sludge

In activated sludge, protozoa feed on free-swimming bacteria and suspended particles, inducing flocculation and increasing the turnover rate of nutrients. In this study, the effect of protozoan grazing on nitrification rates under various conditions in municipal activated sludge batch reactors was examined, as was the spatial distribution of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) within the activated sludge. The reactors were monitored for ammonia, nitrite, nitrate, and total nitrogen concentrations, and bacterial numbers in the presence and absence of cycloheximide (a protozoan inhibitor), allylthiourea (an inhibitor of ammonia oxidation), and EDTA (a deflocculating agent). The accumulations of nitrate, nitrite, and ammonia were lower in batches without than with protozoa grazing. Inhibition of ammonia oxidation also decreased the amount of nitrite and nitrate accumulation. Inhibiting protozoan grazing along with ammonia oxidation further decreased the amounts of nitrite and nitrate accumulated. Induction of deflocculation led to high nitrate accumulation, indicating high levels of nitrification; this effect was lessened in the absence of protozoan grazing. Using fluorescent in situ hybridization and confocal laser scanning microscopy, AOB and NOB were found clustered within the floc, and inhibiting the protozoa, inhibiting ammonia oxidation, or inducing flocculation did not appear to lower the number of AOB and NOB present or affect their position within the floc. These results suggest that the AOB and NOB are present but less active in the absence of protozoa.     

Development of quantitative methods for the detection of enteroviruses in sewage sludges during activation and following land disposal.

The development and evaluation of methods for the quantitative recovery of enteroviruses from sewage sludge are reported. Activated sewage sludge solids were collected by centrifugation, and elution of the solid-associated virus was accomplished by mechanical agitation in glycine buffer at pH 11.0. Eluted viruses were concentrated either onto an aluminum hydroxide floc or by association with a floc which formed de novo upon adjustment of the glycine eluate to pH 3.5. Viruses which remained in the liquid phase after lowering the pH of glycine eluate were concentrated by adsorption to and elution from membrane filters. The method of choice included high pH glycine elution and subsequent low pH concentration; it yielded an efficiency of recovery from activated sludge of 80% for poliovirus type 1, 68% for echovirus type 7, and 75% for coxsackievirus B3. This method was used to study the survival of naturally occurring virus in sludge at a sewage treatment plant and after subsequent land disposal of the solids after aerobic digestion. Reduction of enterovirus titers per gram (dry weight) of solids were modest during sludge activation but increased to a rate of 2 log 10/week after land disposal.     

Development of quantitative methods for the detection of enteroviruses in sewage sludges during activation and following land disposal.

The development and evaluation of methods for the quantitative recovery of enteroviruses from sewage sludge are reported. Activated sewage sludge solids were collected by centrifugation, and elution of the solid-associated virus was accomplished by mechanical agitation in glycine buffer at pH 11.0. Eluted viruses were concentrated either onto an aluminum hydroxide floc or by association with a floc which formed de novo upon adjustment of the glycine eluate to pH 3.5. Viruses which remained in the liquid phase after lowering the pH of glycine eluate were concentrated by adsorption to and elution from membrane filters. The method of choice included high pH glycine elution and subsequent low pH concentration; it yielded an efficiency of recovery from activated sludge of 80% for poliovirus type 1, 68% for echovirus type 7, and 75% for coxsackievirus B3. This method was used to study the survival of naturally occurring virus in sludge at a sewage treatment plant and after subsequent land disposal of the solids after aerobic digestion. Reduction of enterovirus titers per gram (dry weight) of solids were modest during sludge activation but increased to a rate of 2 log 10/week after land disposal.     

Microbial composition and structure of aerobic granular sewage biofilms

Aerobic activated sludge granules are dense, spherical biofilms which can strongly improve purification efficiency and sludge settling in wastewater treatment processes. In this study, the structure and development of different granule types were analyzed. Biofilm samples originated from lab-scale sequencing batch reactors which were operated with malthouse, brewery, and artificial wastewater. Scanning electron microscopy, light microscopy, and confocal laser scanning microscopy together with fluorescence in situ hybridization (FISH) allowed insights into the structure of these biofilms. Microscopic observation revealed that granules consist of bacteria, extracellular polymeric substances (EPS), protozoa and, in some cases, fungi. The biofilm development, starting from an activated sludge floc up to a mature granule, follows three phases. During phase 1, stalked ciliated protozoa of the subclass Peritrichia, e.g., Epistylis spp., settle on activated sludge flocs and build tree-like colonies. The stalks are subsequently colonized by bacteria. During phase 2, the ciliates become completely overgrown by bacteria and die. Thereby, the cellular remnants of ciliates act like a backbone for granule formation. During phase 3, smooth, compact granules are formed which serve as a new substratum for unstalked ciliate swarmers settling on granule surfaces. These mature granules comprise a dense core zone containing bacterial cells and EPS and a loosely structured fringe zone consisting of either ciliates and bacteria or fungi and bacteria. Since granules can grow to a size of up to several millimeters in diameter, we developed and applied a modified FISH protocol for the study of cryosectioned biofilms. This protocol allows the simultaneous detection of bacteria, ciliates, and fungi in and on granules.     

Moderation of ruminal fermentation by ciliated protozoa in cattle fed a high-grain diet.

The objective of this study was to assess the influence of ciliated protozoa on ruminal fermentation in cattle fed high-grain diets. Six ruminally cannulated steers fed a corn-based grain diet (85% concentrate plus 15% alfalfa hay) at 12-h intervals were assigned randomly to two groups, ciliate free and faunated, in a crossover design. Defaunation was by ruminal emptying, omasal flushing, and treatment with sodium sulfosuccinate. Two to 3 weeks after defaunation, the ruminal contents of all steers were sampled before the morning feeding (0 h) and at 1, 2, 4, 6, 8, and 12 h after feeding to measure pH, analyze fermentation products, and monitor counts of ciliated protozoa and lactic acid-producing and -fermenting bacterial groups. Total numbers of ciliated protozoa in the faunated steers averaged 4.3 x 10(5)/g, and the protozoa consisted of nine genera. Ciliate-free steers had lower (P less than 0.01) ruminal pHs (pH 5.97) than faunated cattle (pH 6.45); however, the treatment-time interaction was not significant. Ruminal lactate and ammonia concentrations were similar in both groups. The total volatile fatty acid concentration was higher (P less than 0.05) in the ciliate-free steers than in the faunated steers and exhibited a treatment-time interaction (P less than 0.05). The acetate-to-propionate ratio was higher (P less than 0.05) in the faunated group than in the ciliate-free group and showed a treatment-time interaction (P less than 0.05). Total anaerobic bacterial counts were about fourfold higher in the ciliate-free group than in the faunated group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Moderation of ruminal fermentation by ciliated protozoa in cattle fed a high-grain diet.

The objective of this study was to assess the influence of ciliated protozoa on ruminal fermentation in cattle fed high-grain diets. Six ruminally cannulated steers fed a corn-based grain diet (85% concentrate plus 15% alfalfa hay) at 12-h intervals were assigned randomly to two groups, ciliate free and faunated, in a crossover design. Defaunation was by ruminal emptying, omasal flushing, and treatment with sodium sulfosuccinate. Two to 3 weeks after defaunation, the ruminal contents of all steers were sampled before the morning feeding (0 h) and at 1, 2, 4, 6, 8, and 12 h after feeding to measure pH, analyze fermentation products, and monitor counts of ciliated protozoa and lactic acid-producing and -fermenting bacterial groups. Total numbers of ciliated protozoa in the faunated steers averaged 4.3 x 10(5)/g, and the protozoa consisted of nine genera. Ciliate-free steers had lower (P less than 0.01) ruminal pHs (pH 5.97) than faunated cattle (pH 6.45); however, the treatment-time interaction was not significant. Ruminal lactate and ammonia concentrations were similar in both groups. The total volatile fatty acid concentration was higher (P less than 0.05) in the ciliate-free steers than in the faunated steers and exhibited a treatment-time interaction (P less than 0.05). The acetate-to-propionate ratio was higher (P less than 0.05) in the faunated group than in the ciliate-free group and showed a treatment-time interaction (P less than 0.05). Total anaerobic bacterial counts were about fourfold higher in the ciliate-free group than in the faunated group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Feasibility of using ultrasonic irradiation to recover active biomass from waste activated sludge

Under typical operating conditions, the microbial fraction of activated sludge flocs is approximately 40% by weight. The objective of this research is to evaluate the feasibility of using ultrasonic irradiation to disrupt activated sludge flocs allowing for the subsequent separation of active and inactive fractions. If separation of floc components is possible, then methods may be incorporated into wastewater treatment plant operations whereby only the inactive fraction of floc is wasted (i.e., of waste activated sludge, WAS), which in turn could increase the overall effective biological solids retention time, leading to increased process robustness with no net increase in reactor size. The results indicate that ultrasonic irradiation of WAS at 800 Wl(-1) followed by 30 min of settling can produce a supernatant with heterotrophic specific oxygen uptake rates (SOURs) of over two times the SOUR measured in the bulk mixed liquor. Under these conditions 26% of the initial heterotrophic activity was recovered within only 11% of the initial volatile mass. Similarly, autotrophic analysis revealed that nitrifying organisms, while sensitive to the effects of ultrasonic irradiation, can be separated from the activated sludge floc and recovered. An irradiation density of 200 Wl(-1) with an exposure time between 1 and 2 min produced a supernatant with a specific ammonia removal rate of over two times the initial mixed liquor rate.     

The Removal of Salmonellas in Conventional Sewage Treatment Processes

The numbers of salmonellas in raw sewage entering a treatment plant varied hourly and diurnally; their peak concentration preceded the peak influent waste water flow into the plant by about 2 hours. Salmonellas were detected in all raw influent samples collected from 2 sewage works and the mean population level at the daily peak period was 3000 organisms/1. On average. 70–80% of the salmonellas were removed during primary sedimentation when upwards of 74–84% of solids were removed. Biological treatment and secondary sedimentation removed a further 70–100% of the pathogen. Compared with the activated sludge process the trickling filters were less efficient in removing salmonellas and they were adversely affected by increased loading following rainfall. Considering the whole treatment process, the Guildford works with its activated sludge treatment removed an average of 99–83% salmonellas while the Woking works with its trickling filter plant removed an average of 93–04% of the organisms. The large variance in the numbers of salmonellas in the final effluent from the trickling filters suggest that greater emphasis should be placed on the actual quality of the effluent rather than on percentage removal efficiencies.     

The Removal of Nitrate from Solution by Floc-forming Bacteria on Decomposing Cellulose Particles

S ummary : Cellulose particles in aerated liquid medium inoculated with activated sludge quickly became enveloped in floccular microbial growth (cellulose floc) able to assimilate nitrate rapidly from solution. Sedimenting the floc removed assimilated nitrogen, excess cellulose and biomass. At 18 and 22°, nitrate was removed from solution at 1·76 and 1·83 μg of nitrate-N/ml/h, respectively. Similar results were found with floc formed by a cellulose decomposing isolate and some noncellulolytic floc-forming bacterial contaminants. Washed preformed cellulose floc removed nitrate from dilute solution at 0·89 μg of nitrate-N/ml/h at pH 7·1–8·6. The C : N ratio of the supernatant fluid changed rapidly as nitrate became exhausted; the significance of this is considered in relation to complete removal of C and N by further biological oxidation.     

活性污泥微生物胞外多聚物生物合成途径与菌胶团形成的调控机制

活性污泥法是借助活性污泥微生物菌胶团形成来实现泥水重力分离和部分污泥回用,辅以曝气供氧,在曝气池中高密度的微生物细胞可将溶解性有机污染物迅速降解、转化后为己所用,外排的剩余污泥带走大量有机质和氮磷,水质得以净化。活性污泥微生物所合成的胶质状胞外多聚物(Extracellular polymeric substances,EPS)是污泥菌胶团形成必不可少的"黏合剂",吸水性极高,这也造成剩余污泥难以处置和利用。我们初步总结了活性污泥微生物宏基因组研究概况,利用分子遗传学和基因组学手段,对活性污泥优势种动胶菌(Zoogloea)和其他菌胶团形成菌的EPS生物合成途径和菌胶团形成与调控机制加以研究,鉴定出一个约40 kb的胞外多糖生物合成大型基因簇和一个由7个基因组成的小型基因簇,该基因簇中除胞外多糖合成相关基因外,还编码组氨酸激酶Prs K和反应调节蛋白Prs R双组分系统,可激活RpoNσ因子共同调控一类称之为PEP-CTERM的新型胞外蛋白质的表达,参与菌胶团的形成。PEP-CTERM富含天冬酰胺(缩写为Asn或者N)残基,可能与胞外多糖通过N-连锁的糖基化形成复合物,包裹微生物细胞群体来介导菌胶团的形成。类似的PEP-CTERM基因和胞外多糖合成基因簇在许多重要的活性污泥细菌如聚磷菌和全程氨氧化菌中存在,说明这些细菌也是菌胶团形成菌,可通过污泥沉淀和回用在活性污泥中得以富集。这些研究结果可供活性污泥膨胀控制、污泥减量和剩余污泥资源和能源回收利用参考。     

Metabolic pathways of hydrogen production in fermentative acidogenic microflora

Biohydrogen production from organic wastewater by anaerobically activated sludge fermentation has already been extensively investigated, and it is known that hydrogen can be produced by glucose fermentation through three metabolic pathways, including the oxidative decarboxylation of pyruvic acid to acetyl-CoA, oxidation of NADH to NAD+, and acetogenesis by hydrogen-producing acetogens. However, the exact or dominant pathways of hydrogen production in the anaerobically activated sludge fermentation process have not yet been identified. Thus, a continuous stirred-tank reactor (CSTR) was introduced and a specifically acclimated acidogenic fermentative microflora obtained under certain operation conditions. The hydrogen production activity and potential hydrogen-producing pathways in the acidogenic fermentative microflora were then investigated using batch cultures in Erlenmeyer flasks with a working volume of 500 ml. Based on an initial glucose concentration of 10 g/l, pH 6.0, and a biomass of 1.01 g/l of a mixed liquid volatile suspended solid (MLVSS), 247.7 ml of hydrogen was obtained after a 68 h cultivation period at 35 +/- 1 degrees C. Further tests indicated that 69% of the hydrogen was produced from the oxidative decarboxylation of pyruvic acid, whereas the remaining 31% was from the oxidation of NADH to NAD+. There were no hydrogen-producing acetogens or they were unable to work effectively in the anaerobically activated sludge with a hydraulic retention time (HRT) of less than 8 h.     

BOOK REVIEW

Book reviewed in this article:
Strelkov, A. A., K. M. Sukhanova and I. B. Raikov, eds. 1969. Progress in Protozoology. Abstracts of Papers Read at the IIIrd International Congress on Protozoology, Leningrad, 2–10 July, 1969 .
Curds, C. R. 1969. An illustrated key to the British fresh water ciliated protozoa commonly found in activated sludge .     

A simple method to estimate the contribution of biological floc and reactor-solution to mass transfer of oxygen in activated sludge processes

In this study, the mass transfer coefficient of biological floc (K(L)a(bf)) was estimated from the mass transfer coefficient of the mixed-liquor (K(L)a(f)) and the reactor-solution (K(L)a(e)). The biological floc resistance (BFR) and reactor-solution resistance (SR) were defined as the reciprocal of K(L)a(bf) and K(L)a(e), respectively, by applying the concept of serial-resistance originally presented in two-film theory (Lewis and Whitman (1924) Ind Eng Chem 16:1215-1220). The specific biological floc resistance (SBFR) was defined as biological floc resistance per unit biomass concentration. The data indicated that an activated sludge process yielding low BFR/MLR and BFR/SR tended to produce higher oxygen transfer efficiency. Surprisingly, the reactor-solution posed the same level of resistance as clean water in all experiments, except in a 5-day SRT, non-nitrifying, completely mixed activated sludge (CMAS) process run. Furthermore, SBFR successfully represented biological floc and showed a positive correlation to sludge volume index (SVI). In addition, SBFR/SR and oxygen transfer efficiency (OTE(f)) followed an exponential relationship for the complete data set. The method of separating the mixed-liquor into biological floc and reactor-solution improved the understanding of oxygen transfer under process conditions, without resorting to intrusive techniques or direct handling of fragile biological floc.     

Does a change in reactor loading rate affect activated sludge bioflocculation?

A collection of particles held together by different interparticle forces might eventually give rise to the formation of activated sludge flocs. This process is known as bioflocculation and is crucial for both conventional activated sludge systems and membrane bioreactors. Since industrial wastewater treatment plants generally face varying reactor loading rates due to varying production schemes in the facility, this paper investigates the impact of reactor loading rates on activated sludge bioflocculation. For this purpose, two reactors were initially operated at a nominal reactor loading rate (RLR) and afterwards changed to a high and low RLR. Based on the obtained results, it can be observed that sludge under low RLR conditions is prone to floc fragmentation due to an increase in water-soluble extracellular polymeric substances (EPS). The reactor under high RLR indicated increased floc erosion as a result of increased biomass concentration, which might imply more collisions between sludge flocs, releasing small sludge particles from the floc. In the high RLR reactor, no significant increase in EPS was observed. A distinction between the different (de)flocculation phenomena was made based on sludge volume index, effluent suspended solids and EPS data supplemented with microscopic image analysis.     

Omasal ciliated protozoa in cattle, bison, and sheep.

Omasal contents were collected from slaughtered cattle (n = 54), bison (n = 15), and sheep (n = 40) to determine numbers and generic distribution of ciliated protozoa. Total protozoan numbers were significantly lower in omasal contents than in ruminal contents of all three species, but the percent composition of all protozoan genera was similar between omasal and ruminal populations. The highest numbers of omasal protozoa found were 7.61 X 10(5)/g in cattle, 7.01 X 10(5)/g in bison, and 1.29 X 10(6)/g in sheep. Omasal dry matter was significantly higher than ruminal dry matter in all species and ranged up to 51.5% in cattle fed high-concentrate diets. The omasal pH was similar to the ruminal pH in all species. The number of omasal laminae averaged 149, 145, and 74 for cattle, bison, and sheep, respectively. Although protozoan concentrations in omasal contents were approximately 80% lower than those in ruminal contents, the omasum harbored relatively high numbers of ciliated protozoa. The resident omasal protozoa are extremely difficult to remove, particularly in cattle, and apparently are responsible for reinoculating transiently defaunated rumens.     

Omasal ciliated protozoa in cattle, bison, and sheep

Omasal contents were collected from slaughtered cattle (n = 54), bison (n = 15), and sheep (n = 40) to determine numbers and generic distribution of ciliated protozoa. Total protozoan numbers were significantly lower in omasal contents than in ruminal contents of all three species, but the percent composition of all protozoan genera was similar between omasal and ruminal populations. The highest numbers of omasal protozoa found were 7.61 X 10(5)/g in cattle, 7.01 X 10(5)/g in bison, and 1.29 X 10(6)/g in sheep. Omasal dry matter was significantly higher than ruminal dry matter in all species and ranged up to 51.5% in cattle fed high-concentrate diets. The omasal pH was similar to the ruminal pH in all species. The number of omasal laminae averaged 149, 145, and 74 for cattle, bison, and sheep, respectively. Although protozoan concentrations in omasal contents were approximately 80% lower than those in ruminal contents, the omasum harbored relatively high numbers of ciliated protozoa. The resident omasal protozoa are extremely difficult to remove, particularly in cattle, and apparently are responsible for reinoculating transiently defaunated rumens.     

Clarifying the roles of kinetics and diffusion in activated sludge filamentous bulking

We hypothesized that the growth rates of filaments and floc formers in activated sludge are affected by the combination of kinetic selection (Lou and de los Reyes, Biotechnol Bioeng 92(6): 729-739, 2005b) and substrate diffusion limitation (Martins et al., Water Res 37:2555-2570, 2003). To clarify the influence of these factors in explaining filamentous bulking, a conceptual framework was developed in this study. The framework suggests the existence of three different regions corresponding to bulking, non-bulking, and intermediate regions, based on substrate concentration. In the bulking and non-bulking regions, kinetic growth differences control the competition process, and filaments or floc formers dominate, respectively. In the intermediate region, substrate diffusion limitation, determined by the floc size, plays the major role in causing bulking. To test this framework, sequencing batch reactors (SBRs) were operated with influent COD of 100, 300, 600, and 1,000 mg/L, and the sludge settleability was measured at various floc size distributions that were developed using different mixing strengths. The experimental data in the bulking and intermediate regions supported the proposed framework. A model integrating the two factors was developed to simulate the substrate concentrations at different depths and floc sizes under intermittently feeding conditions. The modeling results confirmed that substrate diffusion limitation occurs inside the flocs at a certain range of activated sludge floc sizes over the operation cycle, and provided additional support for the proposed framework.