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.     

Loss of degradation capacity of activated sludge for a xenobiotic after a period without its influent

Xenobiotic shock experiments were conducted on lab-scale continuous-flow activated sludge systems to examine activated sludge treatment performance and to determine the xenobiotic degrader loss after periods of xenobiotic absence. The systems were operated with normal influent of a xenobiotic and a biogenic substrate until steady state, and were then artificially disturbed by removing and re-adding the xenobiotic in the influent. Substantial xenobiotic leaks were found when xenobiotic absent time was approximately one mean cell residence time (theta(c)), and the system failed when xenobiotic absent time was longer than a theta(c). Amount of degrader at the time of dual substrate steady state was estimated to be approximately 6% of the total sludge. As the xenobiotic absence time was lengthened, degrader amount in the system was reduced exponentially at a half life of approximately three days. The loss rate could be attributed mainly to the rate of displacement by theta(c) operation, followed by endogenous decay and de-acclimation loss.     

Relations between substrate feeding pattern and development of filamentous bacteria in activated sludge processes:

Summary Laboratory scale activated sludge systems were operated under regimes of continuous or intermittent feeding of substrate. In a previous paper it was shown that continuously operated systems resulted in the development of filamentous bacteria and bulking sludges. Intermittently fed sludges resulted in good settling. These results are now confirmed when substrates other than glucose are present in the influent, such as nutrient broth, acetate and starch. With casein deflocculation occurred. For intermittent systems the substrate removal rates were higher than for continuous systems. Based on the results a theory is presented to account for the growth of filamentous bacteria (and bulking) in continuous systems (completely mixed systems). This theory assumes that in intermittently fed systems (plug flow systems) floc forming bacteria become dominant as a result of higher substrate uptake rates and the possibility to survive a starvation phase by thriving on accumulated intracellular metabolites.     

Kinetics of biodegradation of 2,4-dichlorophenoxyacetate in the presence of glucose

Biodegradation of 2,4-dichlorophenoxyacetate (2,4-D) and glucose were studied using a 2,4-D acclimated culture. Biodegradation of glucose by the 2,4-D preacclimated culture follows the typical Monod kinetics after a 20-h lag period in the presence or absence of 2,4-D. Biodegradation of 2,4-D by the same culture follows Andrew's "substrate inhibition" model. When both glucose and 2,4-D are available, mutual inhibition is observed. However, the effect on 2,4-D is masked by the fact that larger concentrations of active biomass are produced when glucose is available. This kind of concurrent utilization and interaction results in a substantial reduction of the mean cell residence time in a continuous flow system. It also extends the area of stability of the process into higher dilution rates as well as into higher influent concentrations.     

Relation between substrate feeding pattern and development of filamentous bacteria in activated sludge processes: Part III. applications with industrial waste waters

Summary Laboratory scale activated sludge systems were operated under regimes of continuous or intermittent feeding of the waste water. Industrial waste waters from breweries, a dairy plant and a petro-chemical plant were investigated. The systems were started up with sludge from a municipal waste water plant or more often with sludges obtained from the corresponding industrial waste water treatment plants. It was found that intermittently fed systems produced sludges with better settleability characteristics than systems that were continuously supplemented with waste water. Our previous hypothesis that in intermittent systems floc forming bacteria become dominant as a result of higher substrate uptake rates was confirmed and may thus be extended to waste waters containing readily available substrates such as carbohydrates (brewery-and dairy waste water) or acids (petro-chemical waste water). Supplementation of brewery waste water with urea had a negative influence on sludge settleability, especially in continuously operated systems.     

Behavior of 2,4-dichlorophenoxyacetic acid degradation and nitrogen conversion by an activated sludge

Summary Degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) was examined together with nitrogen conversion by using an activated sludge acclimated to artificial sewage containing 2,4-D and urea-N, which were the sole carbon and nitrogen sources, respevtively. Ammonification of urea and nitrification of ammonia proceeded concurrently with 2,4-D degradation by the acclimated activated sludge.     

Relations between substrate feeding pattern and development of filamentous bacteria in activated sludge processes

Summary Laboratory scale activated sludge systems were operated under regimes of continuous or intermittent feeding of substrate. It was found that continuously fed systems repeatedly resulted in the development of filamentous bacteria and bulking of the sludge. Intermittently fed systems did form good settling sludges, without filamentous bacteria. The same results were found using different sludge loadings and different concentrations of mixed liquor suspended solids. High dissolved oxygen concentration did not prevent bulking in continuous systems while low dissolved oxygen concentration resulted in bulking with intermittently fed systems. It was found that the substrate removal rate of intermittently operated systems was always higher than for continuously fed systems. The hypothesis is formulated that intermittent feeding leads to higher substrate removal rates by floc forming bacteria and their predominance in intermittently fed systems, which can be compared to plug flow systems.     

Degradation of 2,4-dichlorophenoxyacetic acid by mixed cultures of bacteria

Summary We explored the feasibility of using mixed cultures for herbicide degradation, with the ultimate aim of application for effluent treatment. The present study reports on mixed cultures which were developed to grow aerobically with 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole carbon substrate. Degradation of 2,4-D was verified by HPLC and UV-spectroscopic analysis of the residual 2,4-D concentration in the test cultures. Cultures that were initially developed with 2,4-D also grew readily with glucose, but the degradation of 2,4-D was effectively prevented under mixed substrate conditions. Mamor intermediates or metabolites resulting from 2,4-D degradation were not detected with the HPLC methodology except 2,4-dichlorophenol which appeared to accumulate transiently in the growth medium.     

Pleiotropic Effects of Adaptation to a Single Carbon Source for Growth on Alternative Substrates

It is frequently assumed that populations of genetically modified microorganisms will perform their intended function and then disappear from the environment due to inherent fitness disadvantages resulting from their genetic alteration. However, modified organisms used in bioremediation can be expected to adapt evolutionarily to growth on the anthropogenic substrate that they are intended to degrade. If such adaptation results in improved competitiveness for alternative, naturally occurring substrates, then this will increase the likelihood that the modified organisms will persist in the environment. In this study, bacteria capable of degrading the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) were used to test the effects of evolutionary adaptation to one substrate on fitness during growth on an alternative substrate. Twenty lineages of bacteria were allowed to evolve under abundant resource conditions on either 2,4-D or succinate as their sole carbon source. The competitiveness of each evolved line was then measured relative to that of its ancestor for growth on both substrates. Only three derived lines showed a clear drop in fitness on the alternative substrate after demonstrable adaptation to their selective substrate, while five derived lines showed significant simultaneous increases in fitness on both their selective and alternative substrates. These data demonstrate that adaptation to an anthropogenic substrate can pleiotropically increase competitiveness for an alternative natural substrate and therefore increase the likelihood that a genetically modified organism will persist in the environment.     

Stimulation of methanol degradation in UASB reactors: in situ versus pre-loading cobalt on anaerobic granular sludge

The effect of pre-loading and in situ loading of cobalt onto a cobalt-limited granular sludge on the performance of methanol fed bioreactors was investigated. One upflow anaerobic sludge bed (UASB) reactor was inoculated with cobalt pre-loaded sludge (24h; 30 degrees C; 1 mM CoCl2) and a second UASB with unloaded sludge. The UASB reactors (30 degrees C; pH 7) were operated for 77 days at 8 h hydraulic retention time and organic loading rates ranging from 5 to 20 g COD.L reactor(-1).d(-1). Cobalt pre-loading clearly stimulated the methanogenic activity of the sludge with methanol as the substrate, e.g., after 30 days of reactor operation this activity was 5.8 times higher than that of the cobalt unloaded sludge. During the experiment, part of the cobalt leached from the pre-loaded sludge, i.e., 54% of the cobalt content was lost during the 77 days of reactor operation. Sequential metal extraction showed that losses mainly occurred from the exchangeable and carbonate fraction and in the sludge remaining cobalt was mainly present in the organic/sulfide fraction of the sludge. In situ loading of cobalt in the unloaded UASB reactor on day 57 by adding 31 microM cobalt to the influent for a 24-h period (16% of the cobalt present in the loaded sludge at day 11) resulted in a 4 time increase of the methanogenic activity of the sludge with methanol as the substrate at the end of the reactor experiment, while the accumulated amount of cobalt in the sludge only amounted to 6% of the cobalt accumulated in the loaded sludge (on day 11). This study showed that both pre-loading sludge and in situ loading are adequate for achieving an increased reactor performance of methanol fed UASB reactors operating under cobalt limitation. However, the in situ dosing procedure needs substantially lower amounts of cobalt, while it also gives significantly smaller losses of cobalt with the effluent.     

Adaptation of Delftia acidovorans for degradation of 2,4-dichlorophenoxyacetate in a microfluidic porous medium

Delftia acidovorans MC1071 can productively degrade R-2-(2,4-dichlorophenoxy)propionate (R-2,4-DP) but not 2,4-dichlorophenoxyacetate (2,4-D) herbicides. This work demonstrates adaptation of MC1071 to degrade 2,4-D in a model two-dimensional porous medium (referred to here as a micromodel). Adaptation for 2,4-D degradation in the 2 cm-long micromodel occurred within 35 days of exposure to 2,4-D, as documented by substrate removal. The amount of 2,4-D degradation in the adapted cultures in two replicate micromodels (~10 and 20 % over 142 days) was higher than a theoretical maximum (4 %) predicted using published numerical simulation methods, assuming instantaneous biodegradation and a transverse dispersion coefficient obtained for the same pore structure without biomass present. This suggests that the presence of biomass enhances substrate mixing. Additional evidence for adaptation was provided by operation without R-2,4-DP, where degradation of 2,4-D slowly decreased over 20 days, but was restored almost immediately when R-2,4-DP was again provided. Compared to suspended growth systems, the micromodel system retained the ability to degrade 2,4-D longer in the absence of R-2,4-DP, suggesting slower responses and greater resilience to fluctuations in substrates might be expected in the soil environment than in a chemostat.     

Interdependence between the aerobic degradation of BPA and readily biodegradable substrates by activated sludge in semi-continuous reactors

The objective of the present work was to analyze the interrelationship between the aerobic degradation of BPA and readily biodegradable substrates by activated sludge (AS) in semi-continuous reactors (SCRs). AS were obtained from three SCRs fed with glucose, acetate or peptone. AS from these reactors were used as inocula for three SCRs that were fed with each biogenic substrate, and for three SCRs that were fed with the biogenic substrate and BPA. In all cases, dissolved organic carbon (DOC), BPA, total suspended solids (TSS) and respirometric measurements were performed. Although BPA could be removed in the presence of all the tested substrates, AS grown on acetate exhibited the longest acclimation to BPA. Reactors fed with peptone attained the lowest TSS concentration; however, these AS had the highest specific BPA degradation rate. Specific DOC removal rates and respirometric measurements demonstrated that the presence of BPA had a negligible effect on the removal of the tested substrates. A mathematical model was developed to represent the evolution of TSS and DOC in the SCRs as a function of the operation cycle. Results suggest that the main effect of BPA on AS was to increase the generation of microbial soluble products. This work helps to understand the relationship between the biodegradation of BPA and readily biodegradable substrates.     

Zinc deprivation of methanol fed anaerobic granular sludge bioreactors

The effect of omitting zinc from the influent of mesophilic (30 degrees C) methanol fed upflow anaerobic sludge bed (UASB) reactors, and latter zinc supplementation to the influent to counteract the deprivation, was investigated by coupling the UASB reactor performance to the microbial ecology of the bioreactor sludge. Limitation of the specific methanogenic activity (SMA) on methanol due to the absence of zinc from the influent developed after 137 days of operation. At that day, the SMA in medium with a complete trace metal solution except Zn was 3.4 g CH4-COD g VSS(-1) day(-1), compared to 4.2 g CH4-COD g VSS(-1) day(-1) in a medium with a complete (including zinc) trace metal solution. The methanol removal capacity during these 137 days was 99% and no volatile fatty acids accumulated. Two UASB reactors, inoculated with the zinc-deprived sludge, were operated to study restoration of the zinc limitation by zinc supplementation to the bioreactor influent. In a first reactor, no changes to the operational conditions were made. This resulted in methanol accumulation in the reactor effluent after 12 days of operation, which subsequently induced acetogenic activity 5 days after the methanol accumulation started. Methanogenesis could not be recovered by the continuous addition of 0.5 microM ZnCl2 to the reactor for 13 days. In the second reactor, 0.5 microM ZnCl2 was added from its start-up. Although the reactor stayed 10 days longer methanogenically than the reactor operated without zinc, methanol accumulation was observed in this reactor (up to 1.1 g COD-MeOH L(-1)) as well. This study shows that zinc limitation can induce failure of methanol fed UASB reactors due to acidification, which cannot be restored by resuming the continuous supply of the deprived metal.     

Effects of Adenine Derivatives and Auxin-related Compounds on Mitochondrial Membrane Permeability

Adenine derivatives and auxin-related compounds, 2,4-dichlorophenoxyaceticacid (2,4-D) and 2,3,5-triiodobenzoic acid (TIBA), did not inhibitthe transport systems for succinate or malate into mitochondria.In iso-osmotic KC1 medium, some of these compounds increasedion fluxes moderately. TIBA and 2,4-D inhibited the mitochondrialshrinkage induced by the substrates. In contrast, adenine derivativesinhibited only the shrinkage induced by the substrate whoseoxidation they were able to block specifically. (Received February 18, 1987; Accepted June 29, 1987)     

Dynamics of biodegradation of 2,4-dichlorophenoxyacetate in the presence of glucose

It has been observed experimentally that the biodegradation of 2,4-dichlorophenoxyacetate (2,4-D) is inhibited by the presence of glucose. However, this effect is masked by the fact that larger concentrations of active biomass are produced when glucose is available. The implication of such a "mixed" growth in a continuous flow system is that much higher dilution rates can be applied for an efficient chlorinated-organic removal when other conventional substrates are present. The mean cell residence time is reduced and the area of stability of the process is extended into higher dilution rates, as well as into higher influent concentrations. Finally, the presence of the mixed substrate changes dramatically the "washout" conditions for both substrates. All these facts point out that the biodegradation of chlorinated organics is more efficient in a mixed substrate environment.     

Substrate utilization kinetic model for biological treatment process

The applicability of Contois' kinetic equation to aerobic and anaerobic treatments of organic wastes is investigated. A refractory coefficient to account for the nonbiodegradable portion of the organic substrates in the digester is incorporated into the kinetic equation. The kinetic equation is applied to the data for aerobic digestions of organic substrates and for anaerobic treatment of dairy wastes. They all show a very good fit of the kinetic equation to the data. Furthermore, the kinetic parameters and the refractory coefficients are shown to be independent of influent organic substrate concentration. This study confirms previous reports that the effluent quality of biological treatment systems for organic wastes depends on influent organic waste concentration. The effect of temperature on the kinetic parameters and the refractory coefficient for anaerobic treatment of sewage sludge are studied. It shows that the kinetic parameters vary with temperature, while the refractory coefficient remains fairly constant. Equations to predict biodegradable treatment efficiency and volumetric substrate utilization rate are also briefly discussed.     

Molecular characterization of mesophilic and thermophilic sulfate reducing microbial communities in expanded granular sludge bed (EGSB) reactors

The microbial communities established in mesophilic and thermophilic expanded granular sludge bed reactors operated with sulfate as the electron acceptor were analyzed using 16S rRNA targeted molecular methods, including denaturing gradient gel electrophoresis, cloning, and phylogenetic analysis. Bacterial and archaeal communities were examined over 450 days of operation treating ethanol (thermophilic reactor) or ethanol and later a simulated semiconductor manufacturing wastewater containing citrate, isopropanol, and polyethylene glycol 300 (mesophilic reactor), with and without the addition of copper(II). Analysis, of PCR-amplified 16S rRNA gene fragments using denaturing gradient gel electrophoresis revealed a defined shift in microbial diversity in both reactors following a change in substrate composition (mesophilic reactor) and in temperature of operation from 30°C to 55°C (thermophilic reactor). The addition of copper(II) to the influent of both reactors did not noticeably affect the composition of the bacterial or archaeal communities, which is in agreement with the very low soluble copper concentrations (3–310 μg l⁻¹) present in the reactor contents as a consequence of extensive precipitation of copper with biogenic sulfides. Furthermore, clone library analysis confirmed the phylogenetic diversity of sulfate-reducing consortia in mesophilic and thermophilic sulfidogenic reactors operated with simple substrates.     

The effect of withdrawal of morpholine from the influent and its reinstatement on the performance and microbial ecology of a model activated sludge plant treating a morpholine-containing influent

An activated sludge plant was established which was capable of treating an influent containing morpholine. When this compound was deleted from the influent the ability of the activated sludge to degrade morpholine was reduced. This reduction took the form of an increase in the length of the lag period before morpholine degradation was detected in a die-away test from 0 to ca 1000 h. The decreased ability of the activated sludge to degrade morpholine was accounted for by a decline in the specific population of morpholine-degrading microbes. In this activated sludge all morpholine degraders were Mycobacterium spp. In the absence of morpholine in the influent most mycobacteria in the activated sludge retained their morpholine-degrading phenotypes. This is despite the fact that some of these organisms can lose this phenotype when grown under non-selective conditions. These results are discussed in relation to other work on the degradation of morpholine and to problems in the treatment of xenobiotic compounds in industrial effluents.     

2,4-dichlorophenoxyacetate/alpha-ketoglutarate dioxygenases from Burkholderia cepacia 2a and Ralstonia eutropha JMP134

2,4-Dichlorophenoxyacetate (2,4-D)/alpha-ketoglutarate (alpha-KG) dioxygenase has been purified to apparent homogeneity from Burkholderia cepacia strain 2a, which utilizes 2,4-D as sole carbon source. The enzyme required ferrous ions, and was a homodimer composed of subunits having an Mr of approximately 32,000. The reaction catalysed consumed one mol each of 2,4-D, alpha-KG and dioxygen, with the production of one mol each of succinate, 2,4-dichlorophenol and glyoxylate. Maximum activity was exhibited at pH 7.8 and 25 degrees C, and reactivity was enhanced by the presence of ascorbate and cysteine. Mn2+, Zn2+, Cu2+, Fe3+ and Co2+ were inhibitory, and chemical modification of the dioxygenase revealed that thiol groups were essential for activity. The enzyme was active towards other substituted phenoxyacetates, but reacted most rapidly with 2,4-D. The apparent Michaelis constants for 2,4-D and alpha-KG were 109 and 8.9 microM, respectively. The properties of this enzyme are compared with those of the 2,4-D/alpha-KG dioxygenase from Ralstonia eutropha JMP134, which exhibits a differing N-terminal amino-acid sequence, and a different temperature 'optimum', pH optimum, substrate specificity and sensitivity to thiol-binding reagents.     

Thermophilic (55 degrees C) conversion of methanol in methanogenic-UASB reactors: influence of sulphate on methanol degradation and competition

Two upflow sludge bed reactors (UASB) were operated for 80 days at 55 degrees C with methanol as the substrate with an organic loading rate (OLR) of about 20 g CODl(-1) per day and a hydraulic retention time (HRT) of 10 h. One UASB was operated without sulphate addition (control reactor-R1) whereas the second was fed with sulphate at a COD:SO4(2-) ratio of 10 (sulphate-fed reactor-R2), providing an influent sulphate concentration of 0.6 g l(-1). For both reactors, methanogenesis was the dominant process with no considerable accumulation of acetate. The methanol removal averaged 93% and 83% for R1 and R2, respectively, and total sulphate removal was achieved in the latter. The pathway of methanol conversion for both sludges was investigated by measuring the fate of carbon in the presence and absence of bicarbonate or specific inhibitors for a sludge sample collected at day 72. In both sludges, about 70% of the methanol was syntrophically converted to methane and/or sulphide, via the intermediate H2/CO2. A strong competition between methanogens and sulphidogens took place in the R2 sludge with half of the methanol-COD being used by methane-producing bacteria and the other half by sulphate-reducing bacteria. Acetate was not an important intermediate for both sludges, and played a slightly more important role for the sulphate-adapted sludge (R2), sustained by the higher amount of bicarbonate produced during sulphate-reduction. The pathway study indicates that, although acetate does not represent an important intermediate, the system is susceptible to its accumulation.