Using ATP bioluminescence technique for monitoring microbial activity in sludge

This study demonstrates, for the first time, that the presence of suspended solids in waste‐activated sludge interferes with adenosine triphosphate (ATP) bioluminescence tests. The sludge subject to acid/alkaline treatment represented the test sample. Without consideration of the effect of solid concentrations, one would erroneously estimate the density levels of heterotrophic bacteria in the sludge using ATP data. A light blockage model was proposed to evaluate the luminescence reading without the interference of suspended solids. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 75: 469–474, 2001.     

ATP amplification for ultrasensitive bioluminescence assay: detection of a single bacterial cell

We developed an ultrasensitive bioluminescence assay of ATP by employing (i) adenylate kinase (ADK) for converting AMP + ATP to two molecules of ADP, (ii) polyphosphate (polyP) kinase (PPK) for converting ADP back to ATP (ATP amplification), and (iii) a commercially available firefly luciferase. A highly purified PPK-ADK fusion protein efficiently amplified ATP, resulting in high levels of bioluminescence in the firefly luciferase reaction. The present method, which was approximately 10,000-fold more sensitive to ATP than the conventional bioluminescence assay, allowed us to detect bacterial contamination as low as one colony-forming unit (CFU) of Escherichia coli per assay.     

Homogeneous bioluminescence assay for galactosuria: interference and kinetic analysis

Elevated galactose concentration in urine is an important clinical symptom of galactosemia and other metabolic disorders. A quantitative assay for galactose using firefly luciferase bioluminescence is presented. The assay couples the galactokinase and firefly luciferase reactions. A higher concentration of galactose present in the sample produces a faster decrease in ATP concentration, which is monitored by firefly luciferase bioluminescence. The kinetic assay is modeled and analyzed. The interference between the two reactions, the interference of certain sugars and other components in the urine, the specificity, and the optimal pH for galactokinase were studied. Calibration curves were constructed and compared with a conventional spectrophotometric assay for galactose. The bioluminescence assay is relatively fast and specific for galactose with a linear range from 1 to 20 mM galactose. The effect of other galactose metabolites (galactonate and galactitol) has also been studied.     

Power analysis for real-time PCR quantification of genes in activated sludge and analysis of the variability introduced by DNA extraction

The aims of this study were to determine the power of discrimination of the real-time PCR assay for monitoring fluctuations in microbial populations within activated sludge and to identify sample processing points where methodological changes are needed to minimize the variability in target quantification. DNA was extracted using a commercially available kit from mixed liquor samples taken from the aeration tank of four bench-scale activated-sludge reactors operating at 2-, 5-, 10-, and 20-day solid retention times, with mixed-liquor volatile suspended solid (MLVSS) values ranging from 260 to 2,610 mg/liter. Real-time PCR assays for bacterial and Nitrospira 16S rRNA genes were chosen because they represent, respectively, a highly abundant and a less-abundant bacterial target subject to clustering within the activated sludge matrix. The mean coefficient of variation in DNA yields (measured as microgram of DNA per milligram of MLVSS) in triplicate extractions of 12 different samples was 12.2%. Based on power analyses, the variability associated with DNA extraction had a small impact on the overall variability of the real-time PCR assay. Instead, a larger variability was associated with the PCR assay. The less-abundant target (Nitrospira 16S rRNA gene) had more variability than the highly abundant target (bacterial 16S rRNA gene), and samples from the lower-biomass reactors had more variability than samples from the higher-biomass reactors. Power analysis of real-time PCR assays indicated that three to five samples were necessary to detect a twofold increase in bacterial 16S rRNA genes, whereas three to five samples were required to detect a fivefold increase in Nitrospira 16S rRNA genes.     

Immobilization of activated sludge by PVA-boric acid method

A new method (polyvinyl alcohol-boric acid method) for an inexpensive and effective immobilization of activated sludge was developed. Using activated sludge immobilized by this PVA-boric acid method, synthetic waste-water was treated at a high loading rate of 0.5-2.35 kg TOC/m(3) day. Total organic carbon and total nitrogen were removed at efficiencies of 93 and 30-40%, respectively. The kinetic constants Y and b for this immobilized activated-sludge process were determined to be 0.594g mixed liquor suspended solids (MLSS)/g TOC and 0.0219 day(-1), respectively. The cost calculation of chemicals required for the immobilization of activated sludge by this PVA-boric acid method was proved to be extremely inexpensive for the immobilization of activated sludge.     

Power Analysis for Real-Time PCR Quantification of Genes in Activated Sludge and Analysis of the Variability Introduced by DNA Extraction

The aims of this study were to determine the power of discrimination of the real-time PCR assay for monitoring fluctuations in microbial populations within activated sludge and to identify sample processing points where methodological changes are needed to minimize the variability in target quantification. DNA was extracted using a commercially available kit from mixed liquor samples taken from the aeration tank of four bench-scale activated-sludge reactors operating at 2-, 5-, 10-, and 20-day solid retention times, with mixed-liquor volatile suspended solid (MLVSS) values ranging from 260 to 2,610 mg/liter. Real-time PCR assays for bacterial and Nitrospira 16S rRNA genes were chosen because they represent, respectively, a highly abundant and a less-abundant bacterial target subject to clustering within the activated sludge matrix. The mean coefficient of variation in DNA yields (measured as microgram of DNA per milligram of MLVSS) in triplicate extractions of 12 different samples was 12.2%. Based on power analyses, the variability associated with DNA extraction had a small impact on the overall variability of the real-time PCR assay. Instead, a larger variability was associated with the PCR assay. The less-abundant target (Nitrospira 16S rRNA gene) had more variability than the highly abundant target (bacterial 16S rRNA gene), and samples from the lower-biomass reactors had more variability than samples from the higher-biomass reactors. Power analysis of real-time PCR assays indicated that three to five samples were necessary to detect a twofold increase in bacterial 16S rRNA genes, whereas three to five samples were required to detect a fivefold increase in Nitrospira 16S rRNA genes.     

Enumeration of bacterial cell numbers by amplified firefly bioluminescence without cultivation

We recently developed a novel bioluminescent enzymatic cycling assay for ATP and AMP with the concomitant use of firefly luciferase and pyruvate orthophosphate dikinase (PPDK), where AMP and pyrophosphate produced from ATP by firefly luciferase were converted back into ATP by PPDK. Background luminescence derived from contaminating ATP and AMP in the reagent was reduced using adenosine phosphate deaminase which degrades ATP, ADP, and AMP, resulting in constant and highly amplified bioluminescence with low background luminescence. To detect bacterial cells without cultivation, we applied the above bioluminescent enzymatic cycling reagent to rapid microbe detection system. ATP spots (0.31-5.0 amol/spot) at the level of a single bacterial cell were detected with 5 min signal integration, signifying that integrated luminescence was amplified 43 times in comparison to traditional ATP bioluminescence. Consequently, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Lactobacillus brevis in beer were detected without cultivation. Significant correlation was observed between the number of signal spots obtained using this novel system and the colony-forming units observed with the conventional colony-counting method (R(2)=0.973).     

ATP as a biomarker of viable microorganisms in clean-room facilities

A new firefly luciferase bioluminescence assay method that differentiates free extracellular ATP (dead cells, etc.) from intracellular ATP (viable microbes) was used to determine the viable microbial cleanliness of various clean-room facilities. For comparison, samples were taken from both clean-rooms, where the air was filtered to remove particles >0.5 microm, and ordinary rooms with unfiltered air. The intracellular ATP was determined after enzymatically degrading the sample's free ATP. Also for comparison, cultivable microbial populations were counted on nutrient-rich trypticase soy agar (TSA) plates. Both the cultivable and ATP-based determinations indicate that the microbial burden was lower in clean-room facilities than in ordinary rooms. However, there was no direct correlation between the two sets of measurements because the two assays measured very different populations. A large fraction of the samples yielded no colony formers on TSA, but were positive for intracellular ATP. Subsequently, genomic DNA was isolated directly from selected samples and 16S rDNA fragments were cloned and sequenced, identifying nearest neighbors, many of which are known to be noncultivable in the media employed. It was concluded that viable microbial contamination can be reliably monitored by measurement of intracellular ATP, and that this method may be considered superior to cultivable colony counts due to its speed and its ability to report the presence of viable but noncultivable organisms. When the detection of nonviable microbes is of interest, the ATP assay can be supplemented with DNA analysis.     

ATP生物发光测定试剂研究进展

萤火虫荧光素酶是ATP生物发光试剂的关键组成部分,可通过萤火虫尾提取纯化或基因工程技术制备,酶的活力和纯度决定了ATP生物发光试剂的性能。迄今许多先进技术在ATP生物发光试剂的制备中均有应用,包括酶基因工程改造技术、ATP循环的酶法放大技术、荧光素酶蛋白的活力及发光稳定技术,特异的细胞ATP提取技术等。ATP生物发光试剂的研究焦点主要集中在提高发光试剂的检测灵敏度和性能、增加产品的适应性等方面。     

Rapid separation and quantitation of mixed microorganisms by filtration and bioluminescence

A membrane filtration/bioluminescence system was developed for the differentiation and quantitation of mixed populations of microorganisms. Samples containing microorganisms were filtered through two membrane filters of descending pore size. The microorganisms retained on the filter contain ATP that can be extracted and measured on the filter via the firefly luciferase-luciferin bioluminescence assay. Results, obtained in less than 20 min, show a good correlation (r greater than or equal to 0.95) between the light produced and the number of organisms in the sample. Using these techniques, Escherichia coli can be separated from yeast or mold and measured in samples containing both microorganisms. When lysostaphin is used to selectively lyse Staphylococci on the filter, the specific quantification of these bacteria among other microorganisms can also be accomplished. The filtration/bioluminescence technique offers the potential of being a rapid and sensitive method to differentiate and detect microorganisms, by selective sizing or lysing, in a variety of samples.     

Ozonation strategies to reduce sludge production of a seafood industry WWTP

In this work, several alternatives related to the application of ozone in different streams of a seafood industry WWTP were evaluated to minimize the production of waste sludge. The WWTP was composed of two coagulation-flocculation units and a biological unit and generated around of 6550 kg/d of sludge. Ozone was applied to sludge coming from flotation units (110 g TSS/L) at doses up to 0.03 g O(3)/g TSS during batch tests, no solids solubilization being observed. Ozone doses ranging from 0.007 to 0.02 g O(3)/g TSS were also applied to the raw wastewater in a bubble column reaching a 6.8% of TSS removal for the highest ozone dose. Finally, the effect of the pre-ozonation (0.05 g O(3)/g TSS) of wastewater coming from the first flotation unit was tested in two activated sludge systems during 70 days. Ozonation caused a reduction of the observed yield coefficient of biomass from 0.14 to 0.07g TSS/g COD(Tremoved) and a slight improvement of COD removal efficiencies. On the basis of the capacity for ozone production available in the industry, a maximum reduction of sludge generated by the WWTP of 7.5% could be expected.     

Limitation of sludge biotic index application for control of a wastewater treatment plant working with shock organic and ammonium loadings

This study aimed to determine the relationship between activated sludge microfauna, the sludge biotic index (SBI) and the effluent quality of a full-scale municipal wastewater treatment plant (WWTP) working with shock organic and ammonium loadings caused by periodic wastewater delivery from septic tanks. Irrespective of high/low effluent quality in terms of COD, BOD₅, ammonium and suspended solids, high SBI values (8–10), which correspond to the first quality class of sludge, were observed. High SBI values were connected with abundant taxonomic composition and the domination of crawling ciliates with shelled amoebae and attached ciliates. High SBI values, even at a low effluent quality, limit the usefulness of the index for monitoring the status of an activated sludge system and the effluent quality in municipal WWTP-treated wastewater from septic tanks. It was shown that a more sensitive indicator of effluent quality was a change in the abundance of attached ciliates with a narrow peristome (Vorticella infusionum and Opercularia coarctata), small flagellates and crawling ciliates (Acineria uncinata) feeding on flagellates.     

Enhanced firefly bioluminescence assay of ATP in the presence of ATP extractants by using diethylaminoethyl-dextran

A highly sensitive ATP bioluminescence assay with diethylaminoethyl-dextran (DEAE-Dx) in the presence of ATP extractants such as trichloroacetic acid (TCA) and Triton X-100 is described. These ATP extractants inhibited the activity of firefly luciferase, resulting in a remarkable decrease in the intensity of light emission. However, DEAE-Dx enhanced the intensity of light emission as long as firefly luciferase was active in the presence of the ATP extractants. When DEAE-Dx was used for the assay, the detection limits for ATP in the presence of TCA and Triton X-100 were 0.3 and 0.5 pM, respectively, in aqueous ATP standard solution. The detection limit in the presence of DEAE-Dx was improved 13- to 20-fold compared to that in the absence of DEAE-Dx. The method was applied to the determination of ATP in Escherichia coli extracts. When a 5% solution of TCA was used for the extraction of ATP from E. coli cells, the detection limit corresponded to 250 cells ml(-1) of E. coli.     

The phosphorus fractions and alkaline phosphatase activities in sludge

The alkaline phosphatase activities (APA) and phosphorus fractions in activated sludge during wastewater treatment were studied. Our results showed that the phosphorus concentration and fractions in activated sludge were highly correlated with the characteristics of influents. Inorganic phosphorus (IP) and non-apatite inorganic phosphorus (NAIP) were the main phosphorus fractions of sludge. A larger phosphorus concentration was found in activated sludge due to the more readily mobilizable and bio-available forms. The APA in sludge was directly correlated with mixed liquor suspended solids (MLSS) in activated sludge. The APA in the sludge is implicated the depletion of organic phosphorus forms in sludge, whilst also implying its less inhibition of inorganic phosphorus in sludge. The APA and phosphorus fractions in different sludge samples from the same wastewater treatment plant were quite stable. This stability shows their tight interactions in sludge.     

Viral composition and context in metagenomes from biofilm and suspended growth municipal wastewater treatment plants

Wastewater treatment plants (WWTPs) contain high density and diversity of viruses which can significantly impact microbial communities in aquatic systems. While previous studies have investigated viruses in WWTP samples that have been specifically concentrated for viruses and filtered to exclude bacteria, little is known about viral communities associated with bacterial communities throughout wastewater treatment systems. Additionally, differences in viral composition between attached and suspended growth wastewater treatment bioprocesses are not well characterized. Here, shotgun metagenomics was used to analyse wastewater and biomass from transects through two full-scale WWTPs for viral composition and associations with bacterial hosts. One WWTP used a suspended growth activated sludge bioreactor and the other used a biofilm reactor (trickling filter). Myoviridae, Podoviridae and Siphoviridae were the dominant viral families throughout both WWTPs, which are all from the order Caudovirales. Beta diversity analysis of viral sequences showed that samples clustered significantly both by plant and by specific sampling location. For each WWTP, the overall bacterial community structure was significantly different than community structure of bacterial taxa associated with viral sequences. These findings highlight viral community composition in transects through different WWTPs and provide context for dsDNA viral sequences in bacterial communities from these systems.     

Characterization and biological treatment of ceramic industry wastewater

Ceramic industry wastewaters not only contain high suspended and total solids but also significant amounts of dissolved organics resulting in high BOD or COD loads. Suspended solids can be removed from the wastewater by chemical precipitation. However, dissolved BOD/COD compounds can only be removed by biological or chemical oxidation. Effluent wastewater from chemical sedimentation stage of EGE CERAMIC industry was characterized and subjected to biological treatment in a laboratory scale activated sludge unit. Experiments were conducted at different hydraulic and solids retention times. The best results were obtained with Š_c=20 h of hydraulic and Š_c=20 days of solids retention times (sludge age) resulting in effluent COD concentration of 40 mg/l from a feed wastewater of 720 mg/l COD content. The suspended solids content of the activated sludge effluent was approximately 52 mg/l.     

Dynamic modelling of solids in a full-scale activated sludge plant preceded by CEPT as a preliminary step for micropollutant removal modelling

The presence of micropollutants in the environment has triggered research on quantifying and predicting their fate in wastewater treatment plants (WWTPs). Since the removal of micropollutants is highly related to conventional pollutant removal and affected by hydraulics, aeration, biomass composition and solids concentration, the fate of these conventional pollutants and characteristics must be well predicted before tackling models to predict the fate of micropollutants. In light of this, the current paper presents the dynamic modelling of conventional pollutants undergoing activated sludge treatment using a limited set of additional daily composite data besides the routine data collected at a WWTP over one year. Results showed that as a basis for modelling, the removal of micropollutants, the Bürger–Diehl settler model was found to capture the actual effluent total suspended solids (TSS) concentrations more efficiently than the Takács model by explicitly modelling the overflow boundary. Results also demonstrated that particular attention must be given to characterizing incoming TSS to obtain a representative solids balance in the presence of a chemically enhanced primary treatment, which is key to predict the fate of micropollutants.

     

Effect of cation binding agents on sludge solubilization potential of bacteria

The aim of the present study is to increase sludge solubilization potential of bacteria by the addition of cation binding agents. During the study, three strains of bacteria B1, B2 and B3 were isolated from waste activated sludge acclimatized to a thermophilic condition (55°C). Using these strains the mixed liquor suspended solids degradation was 67, 59, and 33% and the chemical oxygen demand solubilization enhancement was 71, 62, and 36% compared with the control. Cation binding agents such as citric acid, ethylenediaminetetraacetate and sodium tripolyphosphate were added to enhance the sludge solubilization further. Among these, citric acid along with B1 was more effective in solubilization with the mixed liquor suspended solids degradation of 110% and the chemical oxygen demand solubilization enhancement of 115%. 16s rRNA technique was used to identify the bacterial species B1 and it was found to be Bacillus licheniformis. It was also observed that mixed liquor suspended solids reduced rapidly when more soluble chemical oxygen demand was released, thereby increasing sludge solubilization.     

萤火虫的发光行为及其功能起源

发光生物因其活体能自发荧光而倍受关注,萤火虫(鞘翅目:萤科)是被研究最多的发光生物,因为成虫发光在其性选择以及求偶交配中起着重要作用.由于萤火虫的发光是一个消耗能量(ATP),的化学反应过程,因而其发光在成虫之外的虫态也具有重要意义.本文就萤火虫成虫和幼虫的发光行为、功能意义进行了综述,并结合萤火虫的饲养观察对其卵和蛹的发光行为进行了描述,探讨了卵和蛹的形态阶段发光的生物学功能以及生物荧光的起源进化.这将有助于理解萤火虫及其它生物自发荧光的本质和起源进化过程.     

Sorption and precipitation of metals in activated sludge

A conceptual model describing the relative roles of sorption and precipitation processes for metals in solid-solution suspensions is presented. The model performance is demonstrated using experimental data on sorption and precipitation of metals in samples of activated sludge mixed liquor. Based on the experimental results presented here, it appears that, at total metal and mixed liquor suspended solids concentrations and pH values generally encountered in full-scale municipal (or combined municipal/industrial) activated sludge systems, metals are primarily removed by sorption processes.