Detection of sphingomonads and in situ identification in activated sludge using 16S rRNA-targeted oligonucleotide probes

The increasing significance of members of the genus Sphingomonas in biotechnological applications has led to an increased interest in the diversity, abundance and ecophysiological potential of this group of Gram-negative bacteria. This general focus provides a challenge to improve means for identification of sphingomonads; eg molecular genetic methods for rapid and specific detection could facilitate screening of new isolates. Here, fluorescently labeled oligonucleotide probes targeted against 16S rRNA were used to typify strains previously assigned to the genus. All 46 sphingomonads tested including type strains of 21 Sphingomonasspecies could be detected with a probe originally designed for the genus and all but one with a probe designed for the alpha-4 subgroup of the Proteobacteria. The two probes are suitable for direct detection of sphingomonads in pure and mixed cultures as well as in environmental samples of unknown composition. The probes were used to identify sphingomonads in situ in activated sludge samples. Sphingomonads were rather abundant accounting for about 5–10% of the total cells in municipal sludges. Distinct patterns in aggregation of the cells suggest that these organisms could be involved in the formation process of sludge flocs. Received 27 May 1999/ Accepted in revised form 22 August 1999     

利用时间进程法优化活性污泥DG-DGGE图谱

目的:为了探讨电泳时间对双梯度-变性梯度凝胶电泳(DG-DGGE)分析活性污泥样品时的影响。方法:提取污泥DNA后,以通用引物338f/534r扩增16S rDNA序列,采用时间进程法优化PCR扩增产物的DG-DGGE分离效果。结果:采用不同电泳时间进行DGGE分析时,DGGE图谱存在显著的差异。16S rDNA V3区(200 bp)在凝胶梯度6%~12%,变性剂梯度30%~60%时,在200V电压下,最佳电泳时间为5h。     

Occurrence of ammonia-oxidizing Archaea in activated sludges of a laboratory scale reactor and two wastewater treatment plants

Aims:  Characterization of the ammonia-oxidizing archaea (AOA) community in activated sludge from a nitrogen removal bioreactor and wastewater treatment plants (WWTPs).
Methods and Results:  Three primer sets specific for ammonia mono-oxygenase α -subunit ( amoA ) were used to construct clone libraries for activated sludge sample from a nitrogen removal bioreactor. One primer set resulted in strong nonspecific PCR products. The other two clone libraries retrieved both shared and unique AOA amoA sequences. One primer set was chosen to study the AOA communities of activated sludge samples from Shatin and Stanley WWTPs. In total, 18 AOA amoA sequences were recovered and compared to the previous reported sequences. A phylogenetic analysis indicated that sequences found in this study fell into three clusters.
Conclusions:  Different primers resulted in varied AOA communities from the same sample. The AOA found from Hong Kong WWTPs were closely similar to those from sediment and soil, but distinct from those from activated sludge in other places. A comparison of clone libraries between Shatin WWTP and bioreactor indicated the AOA community significantly shifted only after 30-day enrichment.
Significance and Impact of the Study:  This study confirmed the occurrence of AOA in a laboratory scale nitrogen removal bioreactor and Hong Kong WWTPs treating saline or freshwater wastewater. AOA communities found in this study were significantly different from those found in other places. To retrieve diverse AOA communities from environmental samples, a combination of different primers for the amoA gene is needed.     

纳米氧化铈作用下活性污泥胞外聚合物及溶解性微生物产物特性

【目的】纳米氧化铈作为一种应用普遍的人工纳米材料,其生物毒性和环境效应得到了越来越多的重视。尝试从微生物代谢产物的角度,解读纳米氧化铈对活性污泥微生物的影响规律和过程。【方法】在实验室活性污泥系统中投加不同质量浓度纳米氧化铈,研究纳米氧化铈短期作用下微生物胞外聚合物(EPS)和溶解性微生物产物(SMP)这两类主要的微生物代谢产物含量和组分的变化规律。【结果】短期作用下,EPS和SMP的总量都随着纳米氧化铈浓度的增加而增加。低浓度纳米CeO_2不会导致活性污泥中松散型胞外聚合物(LB-EPS)、紧密型胞外聚合物(TB-EPS)含量和组分的显著改变。高浓度纳米CeO_2(25 mg/L以上)作用下TB-EPS含量和组分不受影响,而LB-EPS中多糖和蛋白质为抵抗纳米CeO_2毒性而增多。EPS分层组分含量显著提高,且LB-EPS的增幅显著高于TB-EPS增幅。当纳米氧化铈浓度为50 mg/L时,相较于空白对照组,蛋白质和多糖增幅分别达到35.18%和46.57%。当纳米氧化铈超过25 mg/L以上时,SMP不仅出现蛋白质,多糖和腐殖酸的含量也明显增加。【结论】SMP中蛋白质的产生,可能会与纳米材料相结合,以减小纳米材料的毒性。当纳米氧化铈浓度较低时,EPS的吸附作用会抵制其进入细胞内,当纳米氧化铈浓度较高时,刺激细胞产生更多EPS吸附纳米CeO_2,形成更厚的外部屏障层保护细胞。EPS和SMP的共同作用,构成了微生物细胞对纳米CeO_2的毒性抵抗机制。     

Reducing effect of aerobic selector on the toxicity of synthetic organic compounds in activated sludge process

The effects of phenol, 2-chlorophenol (2-CP), 2,4-dichlorophenol (2,4-DCP) and 1,2,4-trichlorobenzene (1,2,4-TCB) on the biodegradation kinetics of the conventional activated sludge system (CASS) and the selector activated sludge system (SASS) were investigated. Experiments were carried out using a respirometric method on unacclimated biomass from two lab-scale systems that were operated with the sludge age of 8 days. Toxicity of the test compounds for both reactors were arranged according to EC₅₀ (effective concentration) values in order as: 1,2,4-TCB > 2,4-DCP > 2-CP > phenol. All selected test compounds induced higher inhibition effect in the CASS. The SASS appeared to reduce inhibition effect in comparison to the CASS, by 21.36%, 66.95%, 64.37% and 33.33% for phenol, 2-CP, 2,4-DCP and 1,2,4-TCB, respectively. Consequently, the SASS may be recommended as a promising configuration alternative for the waste streams containing toxic compounds.     

Improving the biotreatment of hydrocarbons-contaminated soils by addition of activated sludge taken from the wastewater treatment facilities of an oil refinery

Addition of activated sludge taken from the wastewater treatment facilities ofan oil refinery to a soil contaminated with oily sludge stimulated hydrocarbonbiodegradation in microcosms, bioreactors and biopile. Microcosms containing50 g of soil to which 0.07 % (w/w) of activated sludge was added presented ahigher degradation of alkanes (80 % vs 24 %) and polycyclic aromatic hydrocarbons(PAHs) (77 % vs 49 %) as compared to the one receiving only water, after 30days of incubation at room temperature. Addition of ammonium nitrate or sterilesludge filtrate instead of activated sludge resulted in a similar removal of PAHsbut not of alkanes suggesting that the nitrogen contained in the activated sludgeplays a major role in the degradation of PAHs while microorganisms of thesludge are active against alkanes. Addition of sludge also stimulated hydrocarbonbiodegradation in 10-kg bioreactors operated during 60 days and in a 50-m³ biopile operated during 126 days. This biopile treatment allowed the use of the soil for industrial purpose based on provincial regulation (``C' criteria). In contrast, the soil of the control biopile that received only water still exceeded C criteria for C₁₀–C₅₀ hydrocarbons, total PAHs, chrysene and benzo[a]anthracene.The stimulation effect of sludge was stronger on the 4-rings than on 2-rings PAHs.The soil of the biopile that received sludge was 4–5 times less toxic than the control. These results suggest that this particular type of activated sludge could be used to increase the efficiency of the treatment of hydrocarbon-contaminated soils in a biopile.     

Growth inhibition of activated sludge by humus

Humus at 0.5 to 1 mg l–1 inhibited growth of activated sludge by about 55% in batch and long-term repeated batch cultures without decreasing sugar utilization. The growth inhibition was considerable when concentrations of substrates in the medium supplied per unit weight of activated sludge were low.     

In situ detection of protein-hydrolysing microorganisms in activated sludge

Protein hydrolysis plays an important role in the transformation of organic matter in activated sludge wastewater treatment plants, but no information is currently available regarding the identity and ecophysiology of protein-hydrolysing organisms (PHOs). In this study, fluorescence in situ enzyme staining with casein and bovine serum albumin conjugated with BODIPY dye was applied and optimized to label PHOs in activated sludge plants. A strong fluorescent labeling of the surface of microorganisms expressing protease activity was achieved. Metabolic inhibitors were applied to inhibit the metabolic activity to prevent uptake of the fluorescent hydrolysates by oligopeptide-consuming bacteria. In five full-scale, nutrient-removing activated sludge plants examined, the dominant PHOs were always different morphotypes of filamentous bacteria and the epiflora attached to many of these. The PHOs were identified by FISH using a range of available oligonucleotide probes. The filamentous PHOs belonged to the candidate phylum TM7, the phylum Chloroflexi and the class Betaproteobacteria. In total they comprised 1-5% of the bacterial biovolume. Most of the epiflora-PHOs hybridized with probe SAP-309 targeting Saprospiraceae in the phylum Bacteroidetes and accounted for 8-12% of the total bacterial biovolume in most plants and were thus an important and dominant part of the microbial communities.     

Experimental Approach to the Role of Different Ecological Types of Protozoa in the Activated-sludge System

Relationship between bacteria and ciliates was investigated in continuous cultures with and without the recycling of flocculated biomass. Monocultures of ciliates of various ecological types were used together with mixed cultures of bacteria or bacteria and flagellates. The effect of ciliates on the distribution of flocculated and dispersed, biomass was tested with respect to characterizing an optimum mixed culture for activated-sludge treatment. Monoprotozoal populations were not successful in stabilizing the morphology of a bacterial community in a system with recycling of flocculated biomass. Stabilization was observed (with particular ecological types of ciliates) in the system with a high residence time without recycling and/or in the system with the recycling inoculated by both flagellates and ciliates.     

Examination into the gamma irradiation of activated sludge

This study has shown that the treatment of activated sludge by gamma irradiation resulted in a deterioration in the filterability, a decrease in the size of the floc particles and an increase in the organic matter present in the sludge supernatant. A significant difference was found between the results obtained for filamentous and non-filamentous sludges in relation to the amount of soluble polysaccharide produced.     

Unexpected difference in enantioselective retention on cellulase (CBH I) silica stationary phase caused by exchange of potassium for sodium ion in the mobile phase

An increase in both retention and enantioselectivity for some β-blocking agents was observed when exchanging potassium to sodium ion in the buffer used as mobile phase. A large effect of ionic strength on retention was observed, while the enantioselectivity was constant. Chirality 10:513–518, 1998. © 1998 Wiley-Liss, Inc.     

Sludge minimisation technologies

The treatment and disposal of excess sludge represents a bottleneck of wastewater treatment plants all over the world, due to environmental, economic, social and legal factors. There is therefore a growing interest in developing technologies to reduce the wastewater sludge generation. The goal of this paper is to present the state-of-the-art of current minimisation techniques for reducing sludge production in biological wastewater treatment processes. An overview of the main technologies is given considering three different strategies: The first option is to reduce the production of sludge by introducing in the wastewater treatment stage additional stages with a lower cellular yield coefficient compared to the one corresponding to the activated sludge process (lysis-cryptic growth, uncoupling and maintenance metabolism, predation on bacteria, anaerobic treatment). The second choice is to act on the sludge stage. As anaerobic digestion is the main process in sewage sludge treatment for reducing and stabilising the organic solids, two possibilities can be considered: introducing a pre-treatment process before the anaerobic reaction (physical, chemical or biological pre-treatments), or modifying the digestion configuration (two-stage and temperature-phased anaerobic digestion, anoxic gas flotation). And, finally, the last minimisation strategy is the removal of the sludge generated in the activated sludge plant (incineration, gasification, pyrolysis, wet air oxidation, supercritical water oxidation).     

Salt sensitivity and low discrimination between potassium and sodium in bean plants

Bean plants (Phaseolus vulgaris) were very sensitive to moderate concentrations of NaCl, showing a dramatic decrease in their K⁺ content in the presence of this salt. Increasing the KCl content of the nutrient medium released the inhibitory effect of NaCl by increasing the K⁺ content of the plants. Likewise moderate concentrations of KCl were toxic for bean plants because they produced a large K⁺ loading. NaCl partially released this toxicity by inhibiting the K⁺ loading. When compared to the moderately salt tolerant sunflower plants (Helianthus annuus), bean plants showed a lower capacity to discriminate between K⁺ and Na⁺, at high Na⁺ levels, and an uncontrolled K⁺ uptake at moderate concentrations of K⁺. It is concluded that this low capacity of discrimination of the K⁺ uptake system of bean plants in presence of Na⁺ can account for by the NaCl sensitivity of bean plants.     

Enhancement of the activated sludge process by activated carbon produced from surplus biological sludge

Surplus biological sludge from wastewater treatment operations was converted into activated carbon and then added to the aerated vessel of an activated sludge process treating phenol and glucose. The addition of activated carbon, either sludge-based or commercial, enhanced phenol removal from 58 to 98.7% and from 87 to 93% for COD with feed concentrations of 100 mg phenol l^–1 and 2500 mg COD l^–1. No differences were found between the activated sludge-activated carbon bench scale continuous reactors operating with either commercial or sludge-based activated carbon in spite of the higher adsorption capacity of the former.     

Sodium ion transport in Neurospora crassa

Na⁺ influx and efflux in Neurospora crassa RL21a can be studied separately to calculate net Na⁺ movements. In the absence of external K⁺, Na⁺ influx was independent of the K⁺ content of the cells, but when K⁺ was present, the inhibition of Na⁺ influx by external K⁺ was higher the higher the K⁺ content. Efflux depended on the K⁺ and Na⁺ content, and on the history of the cells. Efflux was higher the higher the Na⁺ and K⁺ contents, and, in low-K⁺ cells, the efflux was also higher in cells grown in the presence of Na⁺ than when Na⁺ was given to cells grown in the absence of Na⁺. Addition of K⁺ to cells in steady state with external Na⁺ resulted in a net Na⁺-loss. In cells grown without Na⁺ this loss was a consequence of the inhibition of Na⁺ influx. In Na⁺-grown cells, addition of K⁺ inhibited Na⁺ influx and increased Na⁺ efflux.     

活性污泥总DNA提取方法的研究

采用冻融＋玻璃珠、溶菌酶＋SDS和冻融＋玻璃珠＋溶菌酶＋SDS的方法提取活性污泥的微生物总DNA,并对以上三种方法进行比较,从中确定最佳的方法,以实现普通实验条件下成功提取符合PCR扩增要求的DNA。经紫外光度分析及电泳结果表明,冻融＋玻璃珠＋溶菌酶＋SDS方法所得的DNA的OD260/OD280为1．81,电泳结果显示,所提DNA片段分子量大于10kb,适于酶解和PCR扩增要求,为PCR技术应用于活性污泥的研究提供了一种简便、可靠的DNA提取方法。