高活性厌氧颗粒污泥稳定性及基质代谢和种间氢转移的研究

选择了几种废水形成的厌氧污泥,进行了稳定性、基质代谢及种间氢转移的研究.颗粒化的污泥对盐、pH、酶作用、温度、压力等外界条件影响有一定的抗性,在丙酸代谢中,丙酸对颗粒污泥抑制浓度可达1000mg/L,而絮状污泥在500mg/L就被明显抑制,并比较了两者的最大比产甲烷速率和氢酶活性,在种间氢转移实验中,乙醇对颗粒污泥的抑制浓度比絮状污泥要高,颗粒污泥在各方面的性能都明显优于絮状污泥,还对颗粒污泥的物理特性及保存方式进行了初步研究.     

Composition and distribution of extracellular polymeric substances in aerobic flocs and granular sludge

Extracellular polymeric substances (EPS) were quantified in flocculent and aerobic granular sludge developed in two sequencing batch reactors with the same shear force but different settling times. Several EPS extraction methods were compared to investigate how different methods affect EPS chemical characterization, and fluorescent stains were used to visualize EPS in intact samples and 20-mum cryosections. Reactor 1 (operated with a 10-min settle) enriched predominantly flocculent sludge with a sludge volume index (SVI) of 120 +/- 12 ml g(-1), and reactor 2 (2-min settle time) formed compact aerobic granules with an SVI of 50 +/- 2 ml g(-1). EPS extraction by using a cation-exchange resin showed that proteins were more dominant than polysaccharides in all samples, and the protein content was 50% more in granular EPS than flocculent EPS. NaOH and heat extraction produced a higher protein and polysaccharide content from cell lysis. In situ EPS staining of granules showed that cells and polysaccharides were localized to the outer edge of granules, whereas the center was comprised mostly of proteins. These observations confirm the chemical extraction data and indicate that granule formation and stability are dependent on a noncellular, protein core. The comparison of EPS methods explains how significant cell lysis and contamination by dead biomass leads to different and opposing conclusions.     

Removal of malachite green (MG) from aqueous solutions by native and heat-treated anaerobic granular sludge

The performance of native and heat-treated anaerobic granular sludge in removing of malachite green (MG) from aqueous solution was investigated with different conditions, such as pH, ionic strength, initial concentration and temperature. The maximum biosorption was both observed at pH 5.0 on the native and heat-treated anaerobic granular sludge. The ionic strength had negative effect on MG removal. Kinetic studies showed that the biosorption process followed pseudo-second-order and q_e for native and heat-treated anaerobic granular sludge is 61.73 and 59.17 mg/g at initial concentration 150 mg/L, respectively. Intraparticle diffusion model could well illuminate adsorption process and faster adsorption rate of native anaerobic granular sludge than heat-treated anaerobic granular sludge. The equilibrium data were analyzed using Langmuir and Freundlich model, and well fitted Langmuir model. The negative values of ΔG° and ΔH° suggested that the interaction of MG adsorbed by native and heat-treated anaerobic granular sludge was spontaneous and exothermic. Desorption studies revealed that MG could be well removed from anaerobic granular sludge by 1% (v/v) of HCl–alcohol solution.     

Dispersal and control of anammox granular sludge at high substrate concentrations

This paper reports about the dispersal and control of anammox granular sludge at high substrate concentrations. The results demonstrate that anammox granular sludge would turn into flocculent sludge when the substrate concentrations exceed the inhibitory threshold concentrations, with an apparent drop in the settling velocity of anammox sludge from 73.73 to 16.49 m/h. Moreover, the sludge was washed out of the reactor and a decrease in the nitrogen removal rate from 23.82 to 16.97 kg N/(m³/day) was observed. The dominant anammox bacteria in the granular and flocculent sludge were Candidatus Kuenenia stuttgartiensis; however, the contents of heme c and extracellular polymeric substances in the flocculent sludge were much lower than in the granular sludge. Furthermore, the chemical composition of extracellular polymeric substances was different. The high nitrite concentrations more than the inhibitory threshold concentrations were regarded as the reason for the observed granular sludge dispersal and deterioration in reactor performance. The apparent dispersed granular sludge and malfunction of reactor performance could be recovered by means of washing out the residual substrate from the reactor and then re-running the reactor from low substrate concentrations.     

Quantification of Methanosaeta Species in Anaerobic Bioreactors Using Genus- and Species-Specific Hybridization Probes

A BSTRACTTo evaluate the role of Methanosaeta spp. in a variety of anaerobic environments, small-subunit rRNA targeted oligonucleotide hybridization probes were developed and experimentally characterized. The probes were designed to be genus specific for Methanosaeta and species specific for Methanosaeta concilii and Methanosaeta thermophila. The temperature of dissociation was determined for each probe. Probe specificities were determined using a diverse collection of Archaea and through an evaluation of probe nesting using samples from a variety of anaerobic bioreactors. Cell fixation and hybridization conditions for fluorescence in situ hybridizations were also evaluated. Although permeability of methanogens was variable, M. concilii cells could be permeabilized using a range of paraformaldehyde and ethanol based fixation conditions. Using the newly designed probes together with previously designed probes for methanogens, it was determined that Methanosaeta spp. were the dominant aceticlastic methanogens in a variety of anaerobic bioreactors when acetate concentrations were low. Their levels were higher in bioreactors with granular sludge than in those with flocculent sludge. In lab-scale upflow anaerobic sludge blanket reactors, the levels of M. concilii rRNA were as high as 30% of the total rRNA.     

Anaerobic degradation of benzoate: batch studies

Response of benzoate along with phenol to different anaerobic inocula has been investigated in batch reactors. In Phase I, the anaerobic biodegradability of benzoate and phenol were evaluated using (a) washed acclimatized granular sludge (WAGS) collected from a passive phenol fed bench-scale up-flow anaerobic sludge blanket reactor (UASBR) and (b) unacclimatized flocculent sludge (UFS) from a UASB based sewage treatment plant (STP). The effect of varying concentrations of benzoate has been investigated in Phase II using acclimatized granular sludge (AGS) from a bench-scale UASBR. Extent of degradation of benzoate was more than the phenol. Increasing benzoate COD from 2500 to 11,700mg/L, resulted in decrease in (i) rate constant, k from 0.79 to 0.11/d and (ii) ultimate biochemical methane potential (microb, g CH4-COD formed/g benzoate COD) from 84% to 60%. Temporal trend conforming to logistic S-curve indicated stressed conditions at higher benzoate concentration. Benzoate degradation was found to be sensitive to nature as well as quantity i.e. food to microorganism (F/M) of inocula used.     

Rapid start-up and performance of denitrifying granular sludge in an upflow sludge blanket (USB) reactor treating high concentration nitrite wastewater

Denitrifying granular sludge reactor holds better nitrogen removal efficiency than other kinds of denitrifying reactors, while this reactor commonly needs seeding anaerobic granular sludge and longer period for start-up in practice, which restricted the application of denitrifying granular sludge reactor. This study presented a rapid and stable start-up method for denitrifying granular sludge. An upflow sludge blanket (USB) reactor with packings was established with flocculent activated sludge for treatment of high concentration nitrite wastewater. Results showed mature denitrifying granular sludge appeared only after 15 days with highest nitrogen removal rate of 5.844 kg N/(m³ day), which was much higher than that of compared anoxic sequencing batch reactor (ASBR). No significant nitrite inhibition occurred in USB and denitrification performance was mainly influenced by hydraulic retention time, influent C/N ratio and internal reflux ratio. Hydraulic shear force created by upflow fluid, shearing of gaseous products and stable microorganisms adhesion on the packings might be the reasons for rapid achievement of granular sludge. Compared to inoculated sludge and ASBR, remarkable microbial communitiy variations were detected in USB. The dominance of Proteobacteria and Bacteroidetes and enrichment of species Pseudomonas_stutzeri should be responsible for the excellent denitrification performance, which further verified the feasibility of start-up method.     

Enhancement of sulfate reduction activity using granular sludge in anaerobic treatment of acid mine drainage

Three laboratory-scale, upflow anaerobic reactors were operated for about 250 d to determine the effect of activated granular sludge with high density of sulfate reducing bacteria in the treatment of artificial acid mine drainage. Sulfate reducing bacteria in the granular sludge taken from the upflow anaerobic sludge blanket reactor were 1–2×10⁶ c.f.u. g^–1, which is at least 10 times higher than that of organic substrates such as cow manure and oak compost. The reactors with granular sludge effectively removed over 99% of heavy metals, such as Fe, Al, Cu, and Cd during the experiment. This result suggests a feasibility of the application of granular sludge as a source of sulfate reducing bacteria for the treatment of acid mine drainage.     

Composition and Distribution of Extracellular Polymeric Substances in Aerobic Flocs and Granular Sludge

Extracellular polymeric substances (EPS) were quantified in flocculent and aerobic granular sludge developed in two sequencing batch reactors with the same shear force but different settling times. Several EPS extraction methods were compared to investigate how different methods affect EPS chemical characterization, and fluorescent stains were used to visualize EPS in intact samples and 20-μm cryosections. Reactor 1 (operated with a 10-min settle) enriched predominantly flocculent sludge with a sludge volume index (SVI) of 120 ± 12 ml g⁻¹, and reactor 2 (2-min settle time) formed compact aerobic granules with an SVI of 50 ± 2 ml g⁻¹. EPS extraction by using a cation-exchange resin showed that proteins were more dominant than polysaccharides in all samples, and the protein content was 50% more in granular EPS than flocculent EPS. NaOH and heat extraction produced a higher protein and polysaccharide content from cell lysis. In situ EPS staining of granules showed that cells and polysaccharides were localized to the outer edge of granules, whereas the center was comprised mostly of proteins. These observations confirm the chemical extraction data and indicate that granule formation and stability are dependent on a noncellular, protein core. The comparison of EPS methods explains how significant cell lysis and contamination by dead biomass leads to different and opposing conclusions.     

Granular sludge growth under different reactor liquid surface tensions in lab-scale upflow anaerobic sludge blanket reactors treating wastewater from sugar-beet processing

The formation of anaerobic granular sludge on wastewater from sugar-beet processing was examined in upflow anaerobic sludge blanket reactors. Two strategies were investigated: addition of high-energy substrate, i.e. sugars, and varying the reactor liquid surface tension. When there were insufficient amounts of sugars i.e. less than 7% of the chemical O₂ demand of the influent, no granulation was observed; moreover lowering the reactor liquid surface tension below 48 mN/m was found to increase biomass wash-out. On the other hand, when there were sufficient sugars, granular sludge growth occurred; moreover operating the reactor at a low reactor liquid surface tension reduced biomass wash-out and increased granular yield.     

厌氧产氢颗粒污泥蛋白质组分析样品的高效制备方法

【背景】厌氧产氢颗粒污泥比絮状产氢污泥具有更高的生物量、沉降性与反应效率,对颗粒污泥进行蛋白质组学研究,有助于揭示其代谢调控的分子机制,从而对厌氧代谢过程进行优化调控。目前关于产氢颗粒污泥蛋白质组分析样品制备方法的研究尚未见文献报道。革兰氏阳性菌Ethanoligenens harbinense YUAN-3是自凝集产氢发酵细菌,在间歇和连续流培养中可形成自聚集的厌氧颗粒,由于其全基因组信息清楚,可作为模式研究材料对制备方法进行评估。【目的】针对厌氧产氢颗粒污泥的蛋白质组学研究,比较不同蛋白质提取方法进行优化。【方法】分别利用液氮研磨、超声破碎、匀浆破碎对产氢颗粒污泥破碎,比较这3种方法对总蛋白提取量的影响;通过双向电泳比较三氯乙酸(Trichloroacetic acid,TCA)-丙酮沉淀法与苯酚抽提法对总蛋白提取效果的影响;对总蛋白样品分别进行同位素标记相对和绝对定量标记(Isobarictagsforrelativeandabsolutequantification,i TRAQ)、串联质谱标签(Tandemmasstag,TMT)标记以及质谱鉴定。【结果】液氮研磨、超声破碎、匀浆破碎3种破碎方法下总蛋白的提取量分别是对照样品的2.0、3.9与5.2倍。与TCA-丙酮沉淀法相比,苯酚抽提法总蛋白样品在双向电泳图谱上的蛋白质点明显增多,分布均匀,同时其在碱性蛋白端与小分子量蛋白端的蛋白质点也明显增多。质谱分析发现,iTRAQ标记样品与TMT标记样品中分别鉴定到1797个与1644个蛋白,在分子量、等电点、亚细胞定位的各个分布范围内,这些蛋白良好地覆盖了E.harbinenseYUAN-3中各个类型的蛋白。【结论】匀浆破碎与苯酚抽提法联用的总蛋白制备方法更适用于厌氧产氢颗粒污泥,该方法有利于后续的蛋白质双向电泳和定量蛋白质组质谱分析,可作为产氢颗粒污泥以及革兰氏阳性菌总蛋白制备的方法参考。     

Organics and nitrogen removal and sludge stability in aerobic granular sludge membrane bioreactor

Novel aerobic granular sludge membrane bioreactor (GMBR) was established by combining aerobic granular sludge technology with membrane bioreactor (MBR). GMBR showed good organics removal and simultaneous nitrification and denitrification (SND) performances for synthesized wastewater. When influent total organic carbon (TOC) was 56.8-132.6 mg/L, the TOC removal of GMBR was 84.7-91.9%. When influent ammonia nitrogen was 28.1-38.4 mg/L, the ammonia nitrogen removal was 85.4-99.7%, and the total nitrogen removal was 41.7-78.4%. Moreover, batch experiments of sludge with different particle size demonstrated that: (1) flocculent sludge under aerobic condition almost have no denitrification capacity, (2) SND capacity was caused by the granular sludge, and (3) the denitrification rate and total nitrogen removal efficiency were enhanced with the increased particle size. In addition, study on the sludge morphology stability in GMBR showed that, although some granular sludge larger than 0.9 mm disaggregated at the beginning of operation, the granular sludge was able to maintain the stability of its granular morphology, and at the end of operation, the amount of granular sludge (larger than 0.18 mm) stabilized in GMBR was more than 56-62% of the total sludge concentration. The partial disaggregation of large granules is closely associated with the change of operating mode from sequencing batch reactor (SBR) system to MBR system.     

Effect of heavy metals on the methanogenic activity of starch-degrading granules

Summary Heavy metals in electroplating effluent inhibited specific methanogenic activity (SMA) of anaerobic starch-degrading granules. The SMA of granules on the degradation of starch were reduced by 50% when each gram of biomass was in contact individually with 105 mg of zinc, 120 mg of nickel, 180 mg of copper, 310 mg of chromium, or >400 mg of cadmium. Granules had higher toxicity-resistance than flocculent sludge, due to their layered structure.     

Phosphorus removal characteristics of granular and flocculent sludge in SBR

Aerobic granulation technology has become a novel biotechnology for wastewater treatment. However, the distinct properties and characteristics of phosphorus removal between granules and flocculent sludge are still sparse in enhanced biological phosphorus removal process. Two identical sequencing batch reactors (SBRs) were operated to compare phosphorus removal performance with granular sludge (R1) and flocculate activated sludge (R2). Results indicated that the start-up period was shorter in R2 than R1 for phosphorus removal, which made R2 reach the steady-state condition on day 21, while R1 was on day 25, and R2 released and took up more phosphorus than R1. As a result, the phosphorus removal was around 90% in R2 while 80% in R1 at the steady-state system. The special phosphorus release rate and special phosphorus uptake rate were 8.818 mg P/g volatile suspended solids (VSS)/h and 9.921 mg P/g VSS/h in R2, which were consistently greater than those (0.999 and 3.016 mg P/g VSS/h) in R1. The chemical oxygen demand removal in two reactors was similar. The granular SBR had better solid-separation performance and higher removal efficiency of NH₄⁺–N than flocculent SBR. Denaturing gradient gel electrophoresis of PCR-amplified 16S rDNA fragment analysis revealed that the diversity and the level of phosphorus-accumulating bacteria in flocculent sludge were much more than those in the granular sludge.     

Anammox反应器启动过程中颗粒污泥性状变化特性

以厌氧颗粒污泥作为接种物,通过185 d的运行,成功启动了上流式厌氧氨氧化污泥床(Upflow anaerobic sludge blanket,UASB)反应器。反应器的进水氨氮与亚硝氮浓度分别提升至224 mg/L和255 mg/L,容积氮去除速率提升至3.76 kg/(m3·d)。采用红外光谱、扫描电镜和透射电镜等对厌氧氨氧化颗粒污泥的性状进行观察,发现颗粒污泥在启动过程中经历了污泥颗粒裂解到污泥颗粒重组的过程,且厌氧氨氧化颗粒污泥表面含有丰富的官能团,说明厌氧氨氧化颗粒污泥可能具有良好的吸附性能。采用宏基因组测序的方法对启动前后颗粒污泥的生态结构进行分析,发现原接种污泥优势菌群(变形菌门、厚壁菌门、拟杆菌门)丰度大幅减少,厌氧氨氧化菌所属的浮霉状菌门丰度则由1.59%提升到23.24%。     

High rate treatment of terephthalic acid production wastewater in a two-stage anaerobic bioreactor

The feasibility was studied of anaerobic treatment of wastewater generated during purified terephthalic acid (PTA) production in two-stage upflow anaerobic sludge blanket (UASB) reactor system. The artificial influent of the system contained the main organic substrates of PTA-wastewater: acetate, benzoate, and terephthalate. Three parallel operated reactors were used for the second stage, and seeded with a suspended terephthalate degrading culture, with and without additional methanogenic granular sludge (two different types). The first stage UASB-reactor was seeded with methanogenic granular sludge. Reactors were operated at 37 degrees C and pH 7. During the first 300 days of operation a clear distinction between the biomass grown in both reactor stages was obtained. In the first stage, acetate and benzoate were degraded at a volumetric loading rate of 40 g-COD/L . day at a COD-removal efficiency of 95% within the first 25 days of operation. No degradation of terephthalate was obtained in the first stage during the first 300 days of operation despite operation of the reactor at a decreased volumetric loading rate with acetate and benzoate of 9 g-COD/L . day from day 150. Batch incubation of biomass from the reactor with terephthalate showed that the lag-phase prior to terephthalate degradation remained largely unchanged, indicating that no net growth of terephthalate degrading biomass occurred in the first stage reactor. From day 300, however, terephthalate degradation was observed in the first stage, and the biomass in this reactor could successfully be enriched with terephthalate degrading biomass, resulting in terephthalate removal capacities of 15 g-COD/L . day. Even though no single reason could be identified why (suddenly) terephthalate degradation was obtained after such a long period of operation, it is suggested that the solid retention time as well the prevailing reactor concentrations acetate and benzoate may have played an important role. From day 1 of operation, terephthalate was degraded in the second stage. In presence of methanogenic granular biomass, high terephthalate removal capacities were obtained in these reactors (15 g-COD/L . day) after approximately 125 days of operation. From the results obtained it is concluded that terephthalate degradation is the bottleneck during anaerobic treatment of PTA-wastewater. Pre-removal of acetate and benzoate in staged bioreactor reduces the lag-phase prior to terephthalate degradation in latter stages, and enables high rate treatment of PTA-wastewater.     

Performance of anaerobic granules for degradation of pentachlorophenol.

Anaerobic granules degrading pentachlorophenol (PCP) with specific PCP removal activity up to 14.6 mg/g of volatile suspended solids per day were developed in a laboratory-scale anaerobic upflow sludge blanket reactor at 28 degrees C, by using a mixture of acetate, propionate, butyrate, and methanol as the carbon source. The reactor was able to treat synthetic wastewater containing 40 to 60 mg of PCP per liter at a volumetric loading rate of up to 90 mg/liter of reactor volume per day, with a hydraulic retention time of 10.8 to 15 h. PCP removal of more than 99% was achieved. Results of adsorption of PCP by granular biomass indicated that the PCP removal by the granules was due to biodegradation rather than adsorption. A radiotracer assay demonstrated that the PCP-degrading granules mineralized [14C]PCP to 14CH4 and 14CO2. Toxicity test results indicated that syntrophic propionate degraders and acetate-utilizing methanogens were more sensitive to PCP than syntrophic butyrate degraders. The PCP-degrading granules also exhibited a higher tolerance to the inhibition caused by PCP for methane production and degradation of acetate, propionate, and butyrate, compared with anaerobic granules unadapted to PCP.     

Unravelling the reasons for disproportion in the ratio of AOB and NOB in aerobic granular sludge

In this study, we analysed the nitrifying microbial community (ammonium-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB)) within three different aerobic granular sludge treatment systems as well as within one flocculent sludge system. Granular samples were taken from one pilot plant run on municipal wastewater as well as from two lab-scale reactors. Fluorescent in situ hybridization (FISH) and quantitative PCR (qPCR) showed that Nitrobacter was the dominant NOB in acetate-fed aerobic granules. In the conventional system, both Nitrospira and Nitrobacter were present in similar amounts. Remarkably, the NOB/AOB ratio in aerobic granular sludge was elevated but not in the conventional treatment plant suggesting that the growth of Nitrobacter within aerobic granular sludge, in particular, was partly uncoupled from the lithotrophic nitrite supply from AOB. This was supported by activity measurements which showed an approximately threefold higher nitrite oxidizing capacity than ammonium oxidizing capacity. Based on these findings, two hypotheses were considered: either Nitrobacter grew mixotrophically by acetate-dependent dissimilatory nitrate reduction (ping-pong effect) or a nitrite oxidation/nitrate reduction loop (nitrite loop) occurred in which denitrifiers reduced nitrate to nitrite supplying additional nitrite for the NOB apart from the AOB.     

Long-term impact of dissolved O(2) on the activity of anaerobic granules

The impact of influent dissolved O(2) on the characteristics of anaerobic granular sludge was investigated at various dissolved O(2) concentrations (0.5-8.1 ppm) in 1- and 5-L laboratory-scale upflow anaerobic sludge bed (UASB)-like anaerobic/aerobic coupled reactors with a synthetic wastewater (carbon sources containing 75% sucrose and 25% acetate). The rate of dissolved O(2) supplied to the coupled reactor was as high as 0.40 g O(2)/L(rx).d, and the anaerobic/aerobic coupled reactors maintained excellent methanogenic performances at a COD loading rate of 3 g COD/L(rx).d even after the reactors had been operated with dissolved O(2) for 3 months. The activities of granular sludge on various substrates (glucose, propionate, and hydrogen) were not impaired, and acetate activity was even improved over a short term. However, after 3 months of operation, slight declines on the acetoclastic activities of granules were observed in the coupled reactor receiving the recirculated fluid containing 8.1 ppm dissolved O(2).Methane yield in the anaerobic control reactor and anaerobic/aerobic coupled reactors revealed that a significant aerobic elimination (up to 30%) of substrate occurred in the coupled reactors, as expected. The presence of dissolved O(2) in the recirculated fluid resulted in the development of fluffy biolayers on the granule surface, which imposed a negative impact on the settleability of granular sludge and caused a slightly higher sludge washout. This research shows that the anaerobic/aerobic coupled reactor can be successfully operated under O(2)-limited conditions and is an ideal engineered ecosystem integrating oxic and anaerobic niches. (c) 1996 John Wiley & Sons, Inc.     

Inhibition of Methanogenesis from Acetate in Granular Sludge by Long-Chain Fatty Acids

The effect of four saturated long-chain fatty acids (caprylic, capric, lauric, and myristic) and one unsaturated long-chain fatty acid (oleic) on the microbial formation of methane from acetate was investigated in batch anaerobic toxicity assays. The tests were carried out with granular sludge from an upflow anaerobic sludge bed reactor. In this sludge, Methanothrix spp. are the predominant acetoclastic methanogens. Lauric acid appeared to be the most versatile inhibitor: inhibition started at 1.6 mM, and at 4.3 mM the maximum specific acetoclastic methanogenic activity had been reduced to 50%. Caprylic acid appeared to be only slightly inhibitory. Oleic acid was almost as inhibitory as lauric acid. Although adsorption of the inhibitor on the cell wall might play an important role in the mechanism of inhibition, the inhibition was found to be correlated with concentration rather than with the amount per unit of biomass. In practical situations, as in anaerobic waste treatment processes, synergism can be expected to enhance the inhibition of methanogenesis. In the present research a background concentration of lauric acid below its MIC strongly enhanced the toxicity of capric acid and (to an even greater extent) myristic acid.