Development of ecotoxicity assay based on inhibition of respiring activity in microbial community using XTT reduction

The aim of this study is to develop ecotoxicity assay for evaluating the influence of chemicals on a microbial ecosystem based on XTT reduction inhibition (XTT assay). XTT reduction method is used for quantification of the microbial respiratory activity. Since the XTT assay indicates the inhibition of microbial respiratory activity, it could evaluate the toxicity of chemicals. Suitable conditions for the XTT assay were determined to be 200 mg/L of particulate organic carbon as test microbe concentration and 15 min of assay time using activated sludge. Toxicities of several chemicals evaluated by activated sludge as test microbes were examined under these conditions. Sensitivity for the toxicity evaluated by the XTT assay using activated sludge microbes was almost the same value was that for the OECD activated sludge respiration inhibition test (ASRI test). XTT assay was also applied for evaluating the influence of chemicals on the soil microbial community and the XTT assay was used to evaluate a median effective concentration (EC(50)) value of 3,5-dichlorophenol (3,5-DCP). The EC(50) value of 3,5-DCP was almost the same as the value using activated sludge as test microbes. These results suggest that the XTT assay using both mixed cultures of non-contaminated environments and chemical extracts from various contaminated environments could evaluate the influence on microbial ecosystems affected by toxic chemicals.     

An Evaluation of Three New-Generation Tetrazolium Salts for the Measurement of Respiratory Activity in Activated Sludge Microorganisms

XTT (3′-[1-[(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonic acid hydrate), MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt), and WST–1 (4-(3-4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio)-1,3-benzenedisulfonate) are tetrazolium salts that have become commercially available only in relatively recent years; they differ from earlier such compounds in that their reduction gives rise to a formazan product that is water soluble. We have established the sites in the prokaryotic respiratory chain at which each of the dyes is reduced to its corresponding formazan and have evaluated the suitability of each for the colorimetric estimation of electron transport system activity in populations of activated sludge microorganisms. Reduction of all three tetrazolium salts was shown to be proportional to cell biomass and oxygen uptake and to be susceptible to low levels of the reference toxicant 3,5-dichlorophenol. XTT, which was not inhibitory at concentrations of up to 2 mM and was reduced by 91% of isolates from a sample of culturable activated sludge bacteria, was chosen for further assay development. XTT-formazan production was found to be stimulated by the availability of an exogenous carbon and energy source, and by the presence of the electron-coupling agent phenazine methosulfate. Less than 3% of XTT reduction by an activated sludge sample was abiotic. An assay based on this compound could be a valuable and simple tool for the routine monitoring of the performance of wastewater treatment systems.     

Diversity of Dominant Bacterial Taxa in Activated Sludge Promotes Functional Resistance following Toxic Shock Loading

Examining the relationship between biodiversity and functional stability (resistance and resilience) of activated sludge bacterial communities following disturbance is an important first step towards developing strategies for the design of robust biological wastewater treatment systems. This study investigates the relationship between functional resistance and biodiversity of dominant bacterial taxa by subjecting activated sludge samples, with different levels of biodiversity, to toxic shock loading with cupric sulfate (Cu[II]), 3,5-dichlorophenol (3,5-DCP), or 4-nitrophenol (4-NP). Respirometric batch experiments were performed to determine the functional resistance of activated sludge bacterial community to the three toxicants. Functional resistance was estimated as the 30 min IC₅₀ or the concentration of toxicant that results in a 50% reduction in oxygen utilization rate compared to a referential state represented by a control receiving no toxicant. Biodiversity of dominant bacterial taxa was assessed using polymerase chain reaction-terminal restriction fragment length polymorphism (PCR-T-RFLP) targeting the 16S ribosomal RNA (16S rRNA) gene. Statistical analysis of 30 min IC₅₀ values and PCR-T-RFLP data showed a significant positive correlation (P < 0.05) between functional resistance and microbial diversity for each of the three toxicants tested. To our knowledge, this is the first study showing a positive correlation between biodiversity of dominant bacterial taxa in activated sludge and functional resistance. In this system, activated sludge bacterial communities with higher biodiversity are functionally more resistant to disturbance caused by toxic shock loading.     

Activated sludge treatment of synthetic wastewater containing pentachlorophenol

Activated sludge treatment of a pentachlorophenol (PCP)-containing synthetic waste was examined. With a waste containing some sugars, and 40-120 mg/L PCP, laboratory activated sludge required about seven days for acclimation. However, the prior addition of a quasipure culture of PCP-metabolizing Arthrobacter resulted in immediate acclimation. Even with acclimated sludge, however, the system was upset for two days by a simple step change from 40 to 120 mg/L of PCP. The stability of the system to such a shock load was considerably improved when a chemostat culture of the PCP-metabolizing Arthrobacter was fed slowly into the mixed liquor. Kinetic models were developed to describe the dynamic response of the system in terms of growth parameters, hydraulic detention time, sludge age, and bleed-in rate from the chemostat.     

Adsorptive immobilization of a Pseudomonas strain on solid carriers for augmented decolourization in a chemostat bioreactor

Pseudomonas GM3, a highly efficient strain in cleavage of azo bonds of synthetic dyes under anoxic conditions, was immobilized via adsorption on two types of carriers, porous glass beads and solid PVA particles. The cells were cultivated in a nutrient medium, adsorbed on sterile carriers, stabilized as biofilms in repeated batch cultures, and introduced into a chemostat activated sludge reactor for augmented decolourization. The microbial cells were quickly adsorbed and fixed on the PVA surface, compared to a slow and linear immobilization on the glass surface. The porous structure of glass beads provided shelter for the embedded cells, giving a high biomass loading or thick biofilm (13.3 mg VS ml-1 carrier) in comparison with PVA particles (4.8 mg VS ml-1 carrier), but the mass transfer of substrate in the biofilm became a significant limiting factorin the thicker biofilms (effectiveness factor eta = 0.31). The microbial decolourization rate per volume of carriers was 0.15 and 0.17 mg dye ml-1 of glass beads and PVA particles, respectively. In augmented decomposition of a recalcitrant azo dye (60 mg l-1), the immobilized Pseudomonas cells in porous glass beads gave a stable decolourization efficiency (80-81%), but cells fixed on solid PVA particles showed an initial high colour removal of 90% which then declined to a stable removal efficiency of 81%. In both cases, the colour removal efficiency of the chemostat bioreactor was increased from < 10% by an activated sludge to approximately 80% by the augmented system.     

Role of anaerobic spore-forming bacteria in the acidogenesis of glucose: Changes induced by discontinuous or low-rate feed supply

A mineral salts medium containing 1% (w/v) glucose providing carbon-limited growth conditions was subjected to anaerobic acidogenesis by mixed populations of bacteria in chemostat cultures. The formation of butyrate was shown to be dependent on the presence of saccharolytic anaerobic sporeformers in the acid-forming population. By the use of pasteurized activated sludge as an inoculum a culture was obtained consisting solely of anaerobic sporeformers that gave rise to the formation of butyrate, acetate, hydrogen and carbon dioxide as the main fermentation products. No formation of propionate could be detected. In this culture, the role of sporulation was investigated by applying periods of starvation and a single-step lowering of dilution rate (shift-down). In an experiment using a mineral salts medium supplemented with 1% (w/v) glucose and 0.5% (w/v) casein hydrolysate formation of refractile forespores as well as cell lysis could be demonstrated after 6 h starvation.In mixed cultures, initially inoculated with non-pasteurized activated sludge, a regular interruption of feed supply for 1 h per day resulted in selection of non-sporulatiog anaerobes. The fermentation pattern changed to a production of propionate and acetate, with a concomitant reduction of gas production. Similar results were obtained with shift-down in dilution rate.     

Application of a Tetrazolium Salt with a Water-Soluble Formazan as an Indicator of Viability in Respiring Bacteria

The tetrazolium salt sodium 3′-{1-[(phenylamino)-carbonyl]-3,4-tetrazolium}-bis (4-methoxy-6-nitro)benzene-sulfonic acid hydrate (XTT) was examined for use as a colorimetric indicator of viability in respiring bacteria. XTT was reduced to an orange, water-soluble formazan product by Methylosinus trichosporium OB3b, Pseudomonas putida, Escherichia coli, and Bacillus subtilis. Formazan production was proportional to live cell biomass, and XTT was reduced by all cultures in the absence of added electron-coupling agents. XTT reduction by M. trichosporium OB3b was linear over several hours and was stimulated by the presence of an exogenous substrate (methanol). Addition of cyanide to cultures incubated under oxic conditions gave an initial 10-fold increase in XTT reduction. Viability of bacteria incubated in the absence of exogenous carbon substrates was measured as XTT reduction and compared with viability estimates from plate counts. Results obtained with the two methods were generally comparable, but the XTT assay was superior when cell recovery on plates was low. Incubation of E. coli for 7 days in the absence of exogenous carbon substrates decreased viability by 90%, whereas the corresponding decreases for cultures of M. trichosporium OB3b, P. putida, and B. subtilis were less than 40%.     

Xenobiotic substrate reduces yield of activated sludge in a continuous flow system

The biomass yield of a continuous flow activated sludge system varied when the system treated influent containing different compositions of biogenic and xenobiotic substrates. Both the biogenic substrate and a test xenobiotic 2,4-dichlorophenoxyacetic acid (2,4-D) were degraded at steady-state activated sludge operations. The true yields, determined from steady-state activated sludge treatment performances, were at the maximum and the minimum when the activated sludge treated the influent of sole biogenic substrate and sole 2,4-D, respectively. The minimum yield was 56% of the maximum. Yield reduction between the maximum and the minimum was proportional to the concentration of 2,4-D in the influent. This trend of yield reduction suited a model that describes the metabolic uncoupling effect of 2,4-D on the sludge's degradation of the substrates. The model function variable was defined as the ratio of 2,4-D to biogenic COD concentrations in the influent.     

Influence of 2,4-dinitrophenol on the characteristics of activated sludge in batch reactors

The response of activated sludge characteristics to the presence of 2,4-dinitrophenol (dNP) in batch cultures was investigated in this study. The sludge yield slightly decreased with an increase in dNP concentration. At 10 mg l(-1), or lower, dNP significantly reduced sludge yield and relative specific growth rates (mu/mu0), but didn't substantially affect its relative specific chemical oxygen demand removal rate (q/q0). Presence of dNP at 1-20 mg l(-1) increased the specific oxygen uptake rate of activated sludge, and slightly changed its hydrophobicity. An analysis on inhibition indicated that the reduction in sludge yield in the presence of dNP was mainly attributed to the significant decreased sludge growth, rather than the reduced substrate degradation.     

Adsorptive Immobilization of a Pseudomonas Strain on Solid Carriers for Augmented Decolourization in a Chemostat Bioreactor

Pseudomonas GM3, a highly efficient strain in cleavage of azo bonds of synthetic dyes under anoxic conditions, was immobilized via adsorption on two types of carriers, porous glass beads and solid PVA particles. The cells were cultivated in a nutrient medium, adsorbed on sterile carriers, stabilized as biofilms in repeated batch cultures, and introduced into a chemostat activated sludge reactor for augmented decolourization. The microbial cells were quickly adsorbed and fixed on the PVA surface, compared to a slow and linear immobilization on the glass surface. The porous structure of glass beads provided shelter for the embedded cells, giving a high biomass loading or thick biofilm (13.3 mg VS ml^?1 carrier) in comparison with PVA particles (4.8 mg VS ml^?1 carrier), but the mass transfer of substrate in the biofilm became a significant limiting factor in the thicker biofilms (effectiveness factor η = 0.31). The microbial decolourization rate per volume of carriers was 0.15 and 0.17 mg dye ml^?1 of glass beads and PVA particles, respectively. In augmented decomposition of a recalcitrant azo dye (60 mg l^?1), the immobilized Pseudomonas cells in porous glass beads gave a stable decolourization efficiency (80 - 81%), but cells fixed on solid PVA particles showed an initial high colour removal of 90% which then declined to a stable removal efficiency of 81%. In both cases, the colour removal efficiency of the chemostat bioreactor was increased from < 10% by an activated sludge to ～80% by the augmented system.     

Development of a test method to assess the sludge reduction potential of aquatic organisms in activated sludge.

This article shows the development of a quantitative sludge reduction test method, which uses the sludge consuming aquatic worm Lumbriculus variegatus (Oligochaeta, Lumbriculidae). Essential for the test are sufficient oxygen supply and the presence of a non-stirred layer of sludge for burrowing of the organisms. The test eliminates the unwanted effects of the organisms' movements, so-called bioturbation, on oxygen transport and (therefore) on sludge reduction. We used fresh untreated activated sludge grown on sewage, in order to stay close to the daily practice of sludge treatment. By separating sludge and worms, sludge reduction and worm growth are quantified independently and accurately. Predation by L. variegatus approximately doubles the decay rate of activated sludge. A minimum ratio of initial worm to sludge biomass (W(0)/S(0)) of about 0.4g worm/g sludge dry mass is required. Under the test conditions 20-40% of the predated sludge is converted into worm biomass. Our test is simple, reproducible and accurate and is done with equipment generally available in any laboratory, yielding results within a few days. The test can also be used to assess the application of mixtures of different aquatic organisms, but does not provide enough information for the design of a sludge treatment reactor.     

Ethanol and lactic acid production from sugar and starch wastes by anaerobic acidification

Anaerobic conversion of carbohydrates can generate various end‐products. Besides physical parameters such as pH and temperature, the types of carbohydrate being fermented influences the fermentation pattern. Under uncontrolled pH, microbial mixed cultures from activated sludge and anaerobic digester sludge anaerobically produced ethanol from glucose while producing lactic acid from starch conversion. This trend was not only observed in batch trials. Also, continuous chemostat operation of anaerobic digester sludge resulted in the reproducible predominance of ethanol fermentation from glucose solution and lactic acid production from starch. Different feeding regimes and substrate availability (shock load versus continuous feeding) in glucose fermentation under non‐controlled pH did not affect the ethanol production as the major end product. Shifts in feed composition from glucose to starch and vice versa result in an immediate change of fermentation end products formation.     

Dynamic effects related to steady-state multiplicity in continuous Saccharomyces cerevisiae cultivations

The behavioral differences between chemostat and productostat cultivation of aerobic glucose-limited Saccharomyces cerevisiae were investigated. Three types of experiments were conducted: a chemostat, where the dilution rate was shifted up or down in stepwise manner; and a productostat, with either stepwise changed or a rampwise increased ethanol setpoint, i.e., an accelero-productostat. The transient responses from chemostat and productostat experiments were interpreted using a simple metabolic flux model. In a productostat it was possible to obtain oxido-reductive steady states at dilution rates far below Dcrit due to a strong repression of the respiratory system. However, these steady states could not be obtained in a chemostat, since a dilution rate shift-down from an oxido-reductive steady state led to a derepression of the respiratory system. It can therefore be concluded that the range of dilution rates where steady-state multiplicity can be obtained differs depending on the operation mode and that this dilution rate multiplicity range may appear larger in a productostat than in a chemostat. A more narrow multiplicity range, however, was obtained when the productostat was operated as an accelero-productostat.     

Quantitation of the Population Size and Metabolic Activity of a Resin Acid Degrading Bacterium in Activated Sludge Using Slot-Blot Hybridization to Measure the rRNA:rDNA Ratio

Abstract The 16S rRNA:rDNA ratio is a useful parameter for measuring metabolic activity of a selected member of a complex microbial community, as in pulp effluent activated sludge systems. The RNA:DNA ratio of Sphingomonas sp. DhA-33, previously isolated from a sequencing batch reactor treating pulp mill effluent, is positively correlated with its growth rate (μ) under steady-state conditions. DhA-33 was grown in a chemostat with growth rates ranging from 0.04 to 0.15 cell divisions per hour. DhA-33 was also able to degrade dehydroabietic acid in bleached kraft mill effluent (BKME) plus mineral medium in batch culture. Slot-blot hybridization with radioactively labeled species-specific oligonucleotide probes for 16S rRNA and 16S rDNA was used to measure rRNA, rDNA, and the RNA:DNA ratio of this strain when in a mixed sludge community. An increase in DhA-33 rDNA indicated growth of DhA-33 within the community. The RNA:DNA ratio of DhA-33 increased sharply during exponential growth and declined as cells entered stationary phase. The RNA:DNA ratio decreased earlier and faster in DhA- 33/sludge co-cultures than in DhA-33 pure cultures, presumably due to an earlier depletion of nutrients. The species-specific quantification of the RNA:DNA ratio makes it possible to estimate the metabolic activity of selected members of a microbial community in situ. Received: 15 March 1999; Accepted: 8 July 1999; Online Publication: 15 February 2000     

Batch biological treatment of nitrogen deficient synthetic wastewater using Azotobacter supplemented activated sludge

Biological treatment of nitrogen deficient wastewaters are usually accomplished by external addition of nitrogen sources to the wastewater which is an extra cost item. As an alternative for effective biological treatment of nitrogen deficient wastewaters, the nitrogen fixing bacterium, Azotobacter vinelandii, was used in activated sludge and also in pure culture. Total organic carbon (TOC) removal performances of Azotobacter-added and free activated sludge cultures were compared at different initial TN/TOC ratios. The rate and extent of TOC removal were comparable for all cultures when initial TN/TOC ratio was larger than 0.12; however, both the rate and extent of TOC removal from nitrogen deficient (TN/TOC<12%) synthetic wastewater were improved by using Azotobacter-added activated sludge as compared to the Azotobacter-free activated sludge culture. More than 90% TOC removal was obtained with pure Azotobacter or Azotobacter-added activated sludge culture from a nitrogen deficient synthetic wastewater.     

Biotransformation of the Major Fungal Metabolite 3,5-Dichloro- p-Anisyl Alcohol under Anaerobic Conditions and Its Role in Formation of Bis(3,5-Dichloro-4-Hydroxyphenyl)methane

Higher fungi have a widespread capacity for biosynthesis of organohalogens. Commonly occurring chloroaromatic fungal metabolites can end up in anaerobic microniches at the boundary of fungal colonies and wetland soils. The aim of this study was to investigate the environmental fate of a major fungal metabolite, 3,5-dichloro-p-anisyl alcohol, under anaerobic conditions. This compound was incubated with methanogenic sludge to study its biotransformation reactions. Initially, 3,5-dichloro-p-anisyl alcohol was readily demethylated in stoichiometric quantities to 3,5-dichloro-4-hydroxybenzyl alcohol. The demethylated product was converted further via two routes: a biotic route leading to the formation of 3,5-dichloro-4-hydroxybenzoate and 2,6-dichlorophenol, as well as an abiotic route leading to the formation of bis(3,5-dichloro-4-hydroxyphenyl)methane. In the first route, the benzyl alcohol moiety on the aromatic ring was oxidized, giving 3,5-dichloro-4-hydroxybenzoate as a transient or accumulating product, depending on the type of methanogenic sludge used. In sludge previously adapted to low-molecular-weight lignin from straw, a part of the 3,5-dichloro-4-hydroxybenzoate was decarboxylated, yielding detectable levels of 2,6-dichlorophenol. In the second route, 3,5-dichloro-4-hydroxybenzyl alcohol dimerized, leading to the formation of a tetrachlorinated bisphenolic compound, which was identified as bis(3,5-dichloro-4-hydroxyphenyl)methane. Since formation of this dimer was also observed in incubations with autoclaved sludge spiked with 3,5-dichloro-4-hydroxybenzyl alcohol, it was concluded that its formation was due to an abiotic process. However, demethylation of the fungal metabolite by biological processes was a prerequisite for dimerization. The most probable reaction mechanism leading to the formation of the tetrachlorinated dimer in the absence of oxygen is presented, and the possible environmental implications of its natural occurrence are discussed.     

Effect of hydraulic regime on organic polymer uptake in the activated sludge process

Mathematical models presented in a previous paper (Biotechnol. Bioeng., 20 , 865 (1978)) describing unsteady-state bacterial growth on organic polymers are modified to predict the holding time necessary to attain a given treatment level in a steady-state activated sludge process. Various hydraulic regimes for the activated sludge aeration basin are assumed, and it shown that the hydraulic regime strongly influences the total holding time necessary to attain a given treatment objective. Maintaining the highest possible concentrations of bacteria, extracelluar enzyme, cleavable polymer bond, and utilizable oligomer in the aeration basin during the course of treatment yields the highest possible organic nutrient uptake. However, it is shown that aeration-basin hydraulic regimes that maintain relatively high levels of bacteria and extracellular enzyme also maintain relatively low levels of polymer bond and utilizable oligomer and vice versa. Thus the efficacy of a particular aeration-basin hydraulic regime depends on its ability to maintain at high levels those concentrations that most strongly influence the kinetics of enzymatic hydrolysis or utilizable oligomer uptake.     

Application of a noninhibitory growth model to predict the transient response in a chemostat

A method of adapting a kinetic model based on steady-state chemostat data to predict the transient performance of a chemostat culture is presented. The proposal provides for a time delay which can be considered equivalent to a period of reduced activity of the organism subsequent to the introduction of a step change in operating conditions. The adapted kinetic model gives substantially better performance in predicting the transient response of an experimental system than the unmodified kinetic model.     

Estimation of steady-state culture characteristics during acceleration-stats with yeasts

Steady-state culture characteristics are usually determined in chemostat cultivations, which are very time-consuming. In contrast, acceleration-stat (A-stat) cultivations in which the dilution rate is continuously changed with a constant acceleration rate are not so time-consuming, especially at high acceleration rates. Therefore, the A-stat could be advantageous to use instead of the chemostat. However, the highest acceleration rate, meaning the fastest A-stat that can be applied for estimating steady-state culture characteristics, is not known yet. Experimental results obtained with Zygosaccharomyces rouxii, an important yeast in soy sauce processes, showed that the culture characteristics during the A-stat with an acceleration rate of 0.001 h(-2) were roughly comparable to those of the chemostat. For higher acceleration rates the deviation between the culture characteristics in the A-stat and those in the chemostat obtained at the same dilution rate generally started to increase. The source of these deviations was examined by simulation for Saccharomyces cerevisiae. The simulations demonstrated that this deviation was not only dependent on the metabolic adaptation rate of the yeast, but also on the rate of change in environmental substrate concentrations during A-stats. From this work, it was concluded that an A-stat with an acceleration rate of 0.001 h(-2) is attractive to be used instead of chemostat whenever a rough estimation of steady-state culture characteristics is acceptable.