Effects of acetate and nitrite addition on fraction of denitrifying phosphate-accumulating organisms and nutrient removal efficiency in anaerobic/aerobic/anoxic process

The effects of acetate and nitrite on the performance of sequencing batch reactors (SBRs) employing an anaerobic/aerobic/anoxic (AOA) process were investigated. Three types of SBR operations were used: sodium acetate addition at the start of anoxic condition for heterotrophic denitrification (Type 1); sodium acetate addition at the start of aerobic condition for anoxic phosphate removal by denitrifying phosphate-accumulating organisms (DNPAOs) (Type 2: conventional AOA process); and nitrite addition at the start of aerobic condition for inhibition of phosphate-accumulating organisms (PAOs) (Type 3). A track experiment shows that Type 2 led to the best performance of SBRs among the three types. An analysis by fluorescence in situ hybridization (FISH) revealed that nitrite addition decreased the ratio of PAOs with a decrease in phosphorus removal efficiency. The fraction of DNPAOs in Type 2 was the highest at 13%, indicating that Type 2 is suitable for the simultaneous nitrogen and phosphorus removal in the AOA process.     

Identification and comparison of aerobic and denitrifying polyphosphate-accumulating organisms

Two laboratory-scale sequencing batch reactors (SBRs) were operated for enhanced biological phosphorus removal (EBPR) in alternating anaerobic-aerobic or alternating anaerobic-anoxic modes, respectively. Polyphosphate-accumulating organisms (PAOs) were enriched in the anaerobic-aerobic SBR and denitrifying PAOs (DPAOs) were enriched in the anaerobic-aerobic SBR. Fluorescence in situ hybridization (FISH) demonstrated that the well-known PAO, "Candidatus Accumulibacter phosphatis" was abundant in both SBRs, and post-FISH chemical staining with 4,6-diamidino-2-phenylindol (DAPI) confirmed that they accumulated polyphosphate. When the anaerobic-anoxic SBR enriched for DPAOs was converted to anaerobic-aerobic operation, aerobic uptake of phosphorus by the resident microbial community occurred immediately. However, when the anaerobic-aerobic SBR enriched for PAOs was exposed to one cycle with anoxic rather than aerobic conditions, a 5-h lag period elapsed before phosphorus uptake proceeded. This anoxic phosphorus-uptake lag phase was not observed in the subsequent anaerobic-aerobic cycle. These results demonstrate that the PAOs that dominated the anaerobic-aerobic SBR biomass were the same organisms as the DPAOs enriched under anaerobic-anoxic conditions.     

Enhancing biological nitrogen removal from tannery effluent by using the efficient Brachymonas denitrificans in pilot plant operations

Summary Laboratory scale and pilot plant reactors were inoculated with an efficient denitrifier, Brachymonas denitrificans(CCUG 45880), in order to evaluate whether a bio-augmentation approach can be used to enhance biological nitrogen removal from tannery effluents. To determine the effectiveness of the introduced strain, denitrifying activity in the activated sludge was monitored by nitrate uptake rate (NUR) measurement of NO₃-N. Fluorescent in situ hybridization (FISH) technique was used to monitor the growth of the augmented species. The laboratory scale nitrate removal efficiency with the introduced B. denitrificans (3.7±0.6 mg NO₃-N gVSS ⁻¹ h ⁻¹) was higher than that of the activated sludge without the addition of the bacteria (3.5±0.7 mg NO₃-N gVSS ⁻¹ h ⁻¹); the NUR in the pilot plant after and before the introduction of the strain was also of the magnitude of 12.0±1.4 and 10.6±1.4 mg NO₃-N gVSS ⁻¹ day ⁻¹ , respectively. In situ hybridization results revealed that the introduced denitrifying bacteria significantly facilitated the development of a dense denitrifying bacterial population in the activated sludge, which enhanced in situ denitrification activity. FISH data indicated that once introduced, B. denitrificans remained abundant throughout the experimental period. The ability to seed a bioreactor with bacterial strain capable of removing target pollutants from tannery effluents in a mixed microbial community suggests that this approach could have commercial applications.     

反硝化聚磷菌及其脱氮除磷机理研究进展

水体富营养化是当前水环境保护工作的重点关注问题,微生物修复富营养化水体具有高效、低耗且不产生二次污染等特点,已经成为富营养化水体生态修复的一种重要方式。近年来,对反硝化聚磷菌的研究及其在污水处理工艺中的应用越来越广泛。不同于传统的反硝化细菌联合聚磷菌去除氮磷工艺,反硝化聚磷菌在交替厌氧、缺氧/好氧条件下能同时进行脱氮除磷而被广泛关注与研究。值得注意的是,近几年报道的部分微生物仅在好氧条件下就可进行同时脱氮除磷,但是其脱氮除磷机理仍未理清。基于此,文中总结了目前发现的反硝化聚磷菌和同时硝化反硝化聚磷微生物的种类及特点,并对其脱氮与除磷的关系及其机理进行了系统性分析,对目前反硝化除磷存在的问题进行了梳理,最后对今后的研究方向进行了展望,以期为完善反硝化聚磷菌的脱氮除磷机理及工艺改进提供参考。     

The fate of phosphate under anoxic conditions in biological nutrient removal activated sludge systems

The nitrogen removal potential of phosphate accumulating organisms under anoxic conditions has been evaluated using a laboratory scale sequencing batch reactor fed with synthetic wastewater and operated in a sequence of anaerobic, anoxic and aerobic periods. The phosphate uptake rate under anoxic conditions was lower than that under aerobic conditions. However, in the presence of an external substrate such as glucose and acetate, the fate of phosphate was dependent on the substrate type; phosphate release occurred in the presence of nitrate as long as acetate was present and glucose did not cause any phosphate release. The nitrate uptake rate was also much lower with glucose than acetate. The results implied that poly-hydroxyalkanoates could be oxidized by nitrate and phosphate uptake during the anoxic phase should be introduced into process modeling. © Rapid Science Ltd. 1998     

Long-term population dynamics and in situ physiology in activated sludge systems with enhanced biological phosphorus removal operated with and without nitrogen removal

Quantitative fluorescence in situ hybridization (FISH) and the combination of FISH with microautoradiography (MAR) were used in order to study the long-term population dynamics (2.5 years) and the in situ physiology in two parallel activated sludge pilot systems with enhanced biological phosphorus removal (EBPR). The two systems received the same influent wastewater, but were differently operated (with and without nitrogen removal, respectively). Both systems showed a significant P removal that increased when different substrates (phosphorus (P), acetate and glucose, respectively) were added to the influent wastewater. Rhodocyclus-related bacteria were present in both systems in significant numbers (ranging from 4 to 28%) throughout the whole period. This supports the hypothesis that these bacteria occur in significant numbers in different types of well-operating EBPR activated sludge processes. However, we observed a lower correlation (< 0.5) for the amount of Rhodocyclus-related bacteria to the P content in activated sludge than previous studies (> 0.9). The Actinobacteria were the only additional group of bacteria which showed a similar degree of correlation to the P content in activated sludge as the Rhodocyclus-related bacteria--but only for the system without nitrogen removal. Significant amounts (< or = 12%) of glycogen-accumulating bacteria (GAOs) were detected in the system with nitrogen removal (but not in the other system), but had no, in contrast to previous observations, apparent negative effect on the overall EBPR performance. FISH-MAR indicated that a significant part of the Betaproteobacteria (part of them identified as Rhodocyclus-related bacteria) as well as the Actinobacteria were able to take up 33Pi, [3H]-acetate and [3H]-glucose under anaerobic-aerobic conditions. The contribution of anoxic 33Pi uptake under alternating anaerobic-anoxic conditions was significantly lower. Interestingly, not all Rhodocyclus-related bacteria showed uptake of these three radioactive substrates. This may be due to differences in metabolic state, physiological potential or genotype, not detectable by the present probe set for Rhodocyclus-related bacteria. Comparison of the 33Pi, [3H]-acetate and [3H]-glucose uptake by activated sludge after different fixation and incubation procedures showed that a part of the observed 33Pi uptake may have been caused by a combination of a biological and chemical or biologically induced chemical P adsorption.     

Biomass granulation in an aerobic:anaerobic-enhanced biological phosphorus removal process in a sequencing batch reactor with varying pH

Long-term influences of different steady-state pH conditions on microbial community composition were determined by fluorescence in situ hybridization (FISH) in a laboratory scale reactor configured for enhanced biological phosphorus removal (EBPR). Chemical profiles were consistent with shifts in populations from polyphosphate-accumulating organisms (PAO) to glycogen-accumulating organisms (GAO) when pH fell from pH 7.5 to 7.0 and then to 6.5. While biomass was both dispersed and flocculated at pH 7.5, almost complete granulation occurred gradually after pH was dropped to 7.0, and these granules increased in size as the pH was reduced further to 6.5. Reverting back to pH 7.5 led to granule breakdown and corresponding increases in anaerobic phosphate release. Granules consisted almost entirely of Accumulibacter PAO cells, while putative GAO populations were always present in small numbers. Results suggest that low pH may contribute to granulation under these operational conditions. While chemical profiles suggested the PAO:GAO balance was changing as pH fell, FISH failed to reveal any marked corresponding increase in GAO abundances. Instead, TEM evidence suggested the Accumulibacter PAO phenotype was becoming more like that of a GAO. These data show how metabolically adaptable the Accumulibacter PAO can be under anaerobic:aerobic conditions in being able to cope with marked changes in plant conditions. They suggest that decreases in EBPR capacity may not necessarily reflect shifts in community composition, but in the existing population metabolism.     

Enhanced biological phosphate removal by granular sludge in a sequencing batch reactor

A laboratory-scale sequencing batch reactor was started-up with flocculated biomass and operated primarily for enhanced biological phosphate removal. Ten weeks after the start-up, gradual formation of granular sludge was observed. The compact biomass structure allowed halving the settling time, the initial reactor volume, and doubling the influent COD concentration. Continued operation confirmed the possibility of maintaining a stable granular biomass with a sludge volume index less than 40 ml g^–1, while securing a removal efficiency of 95% for carbon, 99.6% for phosphate, and 71% for nitrogen. Microscopic observations revealed a morphological diversity.     

Ecophysiology of polyphosphate-accumulating organisms and glycogen-accumulating organisms in a continuously aerated enhanced biological phosphorus removal process

Aims: To investigate the ecophysiology of populations of polyphosphate-accumulating organisms (PAO) and glycogen-accumulating organisms (GAO) in communities of a novel acetate fed process removing phosphate from wastewater. Attempts were made to see if acetate could be replaced by an alternative carbon source which did not support the growth of the GAO. Methods and Results: A continuously aerated sequencing batch reactor was operated with different acetate feed levels. Fluorescence in situ hybridization (FISH) showed that Defluviicoccus GAO numbers increased at lower acetate feed levels. With FISH/microautoradiography (MAR) both detected morphotypes of Defluviicoccus assimilated a wider range of substrates aerobically than Accumulibacter PAO. Their uptake profile differed from that reported for the same phylotype in full scale anaerobic : aerobic EBPR plants. Conclusions: This suggests that replacing acetate with another substrate is unlikely to provide Accumulibacter with a selective advantage in this process. Why Defluviicoccus appeared to out-compete Accumulibacter at lower acetate concentrations was not clear. Data suggest physiological and morphological diversity may exist within a single Defluviicoccus phylotype. Significance and Impact of the Study: This study implies that the current FISH probes for Defluviicoccus GAO may not reveal the full extent of their biodiversity, and that more information is required before strategies for their control can be devised.     

Anaerobic metabolism of propionate by polyphosphate-accumulating organisms in enhanced biological phosphorus removal systems

Propionate, a carbon substrate abundant in many prefermenters, has been shown in several previous studies to be a more favorable substrate than acetate for enhanced biological phosphorus removal (EBPR). The anaerobic metabolism of propionate by polyphosphate accumulating organisms (PAOs) is studied in this paper. A metabolic model is proposed to characterize the anaerobic biochemical transformations of propionate uptake by PAOs. The model is demonstrated to predict very well the experimental data from a PAO culture enriched in a laboratory-scale reactor with propionate as the sole carbon source. Quantitative fluorescence in-situ hybridization (FISH) analysis shows that Candidatus Accumulibacter phosphatis, the only identified PAO to date, constitute 63% of the bacterial population in this culture. Unlike the anaerobic metabolism of acetate by PAOs, which induces mainly poly-beta-hydroxybutyrate (PHB) production, the major fractions of poly-beta-hydroxyalkanoate (PHA) produced with propionate as the carbon source are poly-beta-hydroxyvalerate (PHV) and poly-beta-hydroxy-2-methylvalerate (PH2MV). PHA formation correlates very well with a selective (or nonrandom) condensation of acetyl-CoA and propionyl-CoA molecules. The maximum specific propionate uptake rate by PAOs found in this study is 0.18 C-mol/C-mol-biomass . h, which is very similar to the maximum specific acetate uptake rate reported in literature. The energy required for transporting 1 carbon-mole of propionate across the PAO cell membrane is also determined to be similar to the transportation of 1 carbon-mole of acetate. Furthermore, the experimental results suggest that PAOs possess a similar preference toward acetate and propionate uptake on a carbon-mole basis.     

Transformation of phosphorus and relevant intracellular compounds by a phosphorus-accumulating enrichment culture in the presence of both the electron acceptor and electron donor

This study was conducted to obtain a better insight into the metabolic behavior of denitrifying phosphate-accumulating organisms relative to the transformations of relevant intracellular compounds as well as phosphorus and nitrate for enhanced biological phosphorus removal under different combinations of electron acceptor (oxygen or nitrate) and electron donor (acetate). Under anoxic conditions, the amount of polyhydroxybutyrate (PHB) produced per acetate taken up considerably increased with the increasing amount of nitrate reduced whereas the amounts of nitrate reduced and phosphorus released per acetate taken up remained almost constant. However, glycogen utilization occurred during PHB production and then was again observed in response to the initial supplementation of acetate after glycogen accumulation was transiently observed during anoxic phosphorus uptake using nitrate as an electron acceptor. On the other hand, under subsequent aerobic conditions, the additional supplementation of acetate again caused aerobic phosphorus release and PHB production, which showed that PHB production was associated with polyphosphate cleavage regardless of electron acceptor conditions. In contrast to anoxic conditions, glycogen accumulation was observed during PHB production. Based on these observations, the preliminary model for the metabolic behavior of denitrifying phosphate-accumulating organisms was proposed and could well account for the complex transformations of PHB and glycogen together with phosphorus release in the presence of acetate under different electron acceptors.     

Role of nitrate in biological phosphorus removal in a sequencing batch reactor

Summary The effects of nitrate on phosphorus release and uptake in a sequencing batch reactor for biological phosphorus removal was investigated. The addition of nitrate decreased phosphorus release in the anaerobic stage. The synthesis of poly(hydroxyalkanoates) was decreased with the presence of nitrate, possibly due to the competitive utilization of the carbon source by PHA synthesis and denitrification of nitrate. Instead of oxygen, nitrate could be used as an electron acceptor for phosphorus removal. However, the simultaneous addition of nitrate and acetate greatly reduced the phosphorus removal rate. Phosphate and nitrate could be removed simultaneously with nitrate as the electron acceptor, and the continuous and steady feeding of nitrate was beneficial to phosphate removal.     

Enhanced biological phosphorus removal in a sequencing batch reactor using propionate as the sole carbon source

An enhanced biological phosphorus removal (EBPR) system was developed in a sequencing batch reactor (SBR) using propionate as the sole carbon source. The microbial community was followed using fluorescence in situ hybridization (FISH) techniques and Candidatus 'Accumulibacter phosphatis' were quantified from the start up of the reactor until steady state. A series of SBR cycle studies was performed when 55% of the SBR biomass was Accumulibacter, a confirmed polyphosphate accumulating organism (PAO) and when Candidatus 'Competibacter phosphatis', a confirmed glycogen-accumulating organism (GAO), was essentially undetectable. These experiments evaluated two different carbon sources (propionate and acetate), and in every case, two different P-release rates were detected. The highest rate took place while there was volatile fatty acid (VFA) in the mixed liquor, and after the VFA was depleted a second P-release rate was observed. This second rate was very similar to the one detected in experiments performed without added VFA.A kinetic and stoichiometric model developed as a modification of Activated Sludge Model 2 (ASM2) including glycogen economy, was fitted to the experimental profiles. The validation and calibration of this model was carried out with the cycle study experiments performed using both VFAs. The effect of pH from 6.5 to 8.0 on anaerobic P-release and VFA-uptake and aerobic P-uptake was also studied using propionate. The optimal overall working pH was around 7.5. This is the first study of the microbial community involved in EBPR developed with propionate as a sole carbon source along with detailed process performance investigations of the propionate-utilizing PAOs.     

Enhanced enzymatic synthesis of a semi-synthetic cephalosprin,cefaclor, with in situ product removal

In the enzymatic synthesis of cefaclor, 3-chloro-7-d-(2-phenylglycinamide)-3-cephem-4-carboxylic acid, from phenylglycine methyl ester and 7-aminodesacetoxymethyl-3-chlorocephalosporanic acid, the in situ product could influence both the overall conversion and hydrolysis of the ester. Optimization of the parameters, such as pH 6.2, 5 °C and substrate molar ratio of 2:1, made in situ product removal improve the overall conversion from 64% to 85% (mol/mol).     

Characterizing biological aerosol in a chamber: an approach to estimation of viable organisms in a single biological particle

There are practical and valid reasons topresent biological field trial referenceresults as agent containing particles per literof air (ACPLA). However, workers in biologicalaerosol research have a need to know how manyviable individual organisms are contained in asingle particle of a given diameter. Anecdotalevidence may exist suggesting that the task hasbeen accomplished but without a way toreplicate the measurements, it is difficult toaccept unsubstantiated claims. It is verydifficult to declare a finite number thatsatisfies all the experimental requirements, asthe problem is a statistical probability issue. This paper describes a method for estimatingthe number with practical instructions forreplicating the work in other laboratories.The test aerosol was contained in a 90 m³chamber at concentrations as low as 5 ACPLA. Amodified version of slit sampler collectedviable particles. A statistical method was usedto demonstrate sampling predictability at 95%confidence level. By using glass fiber filtersmounted in a dichotomous sampler, samplingefficiencies were estimated for a variety ofcommon aerosol collectors. The accumulated datapermitted the estimation of the number ofviable spores per particle. For a 2.5 to4 µm particle, arguments have beenpresented for considering 4.5 as the mostprobable ACPLA value.     

Comparative analysis of biological phosphate removal (BPR) and non-BPR activated sludge bacterial communities with particular reference to Acinetobacter

The bacterial community of a biological phosphate removal (BPR) activated sludge process was studied and compared to that of a non-BPR process treating the same municipal waste water. Bacterial isolates from the BPR process, as characterized by whole cell fatty acids, belonged to more than twenty genera, with Micrococcus, Staphylococcus and Acidovorax scoring highest. Acinetobacter spp represented 4% of cultured bacteria, ≤3% as estimated by fluorescence in situ hybridization, and well under 10% on the basis of the proportion of ubiquinone Q9 in the sludge. The mole proportions of ubiquinones, Q8 : Q10 : Q9 in the sludge were maintained fairly stable at approximately 9:4:1. The spectra of the isolated strains and the proportions of ubiquinones in the processes (BPR vs non-BPR) were otherwise similar, but a significant number of isolates related to actinomycetes were obtained from the BPR sludge only. The BPR process did not enrich Acinetobacter. Pure cultures of Acinetobacter isolated from the sludge stained for polyphosphate, but Acinetobacter cells responding to the ACA probe in native sludge from the BPR process did not. Instead, the bulk of the polyphosphate in the BPR sludge was located in a distinct morphotype of large, coccoid, highly clustered cells. Received 3 August 1998/ Accepted in revised form 29 October 1998     

人单拷贝及重复DNA序列的原位PCR

在培养的人小肠癌转移腹水细胞系细胞中进行了Y染色体特异的重复序列及单拷贝序列的原位扩增与检测.结果显示原位PCR法的灵敏度比直接的原位杂交法明显提高.     

Effect of pH change on the performance and microbial community of enhanced biological phosphate removal process

An acetate-rich wastewater, containing 170 mg/L of total organic carbon (TOC), 13 mg/L of N, and 15 mg/L of P, was treated using the enhanced biological phosphate removal (EBPR) process operated in a sequencing batch reactor. A slight change of pH of the mixed liquor from 7.0 to 6.5 led to a complete loss of phosphate-removing capability and a drastic change of microbial populations. The process steadily removed 94% of TOC and 99.9% of P from the wastewater at pH 7.0, but only 93% TOC and 17% of P 14 days after the pH was lowered to pH 6.5. The sludge contained 8.8% P at pH 7.0, but only 1.9% at pH 6.5. Based on 16S rDNA analysis, 64.8% of the clones obtained from the sludge at pH 7.0 were absent in the pH 6.5 sludge. The missing microbes, some of which were likely responsible for the phosphate removal at pH 7.0, included beta-Proteobacteria, Actinobacteria, Bacteriodetes/Chlorobi group, plus photosynthetic bacteria and Defluvicoccus of the alpha-Proteobacteria. Among them, the last two groups, which represented 9.3% and 10.1% of the EBPR sludge at pH 7.0, have rarely been reported in an EBPR system.     

Isolation and Characterization of a Satellite DNA Family in Achirus lineatus (Teleostei: Pleuronectiformes: Achiridae)

Agarose gels stained with Ethidium bromide and Southern blot experiments of HindIII-digested genomic DNA of Achirus lineatus evidenced the presence of monomers and multimers of a DNA segment of about 200 bp, named here Al-HindIII sequence. No signals were observed in Southern blot experiments with genomic DNA of other flatfish species. The DNA sequencing of four recombinant clones showed that Al-HindIII sequences had 204 bp and were 63.72% AT-rich. FISH experiments using a Al-HindIII sequence as probe showed bright signals in the centromeric position of all chromosomes of A. lineatus.