Biodegradation of N-Methylmorpholine-N-oxide

N-Methylmorpholine-N-oxide (NMMO) is capable of dissolving cellulose without any further addition of chemicals. The solution can be used to produce cellulosic staple fibres by pressing it through spinning jets into an aqueous spinning bath. Because of results from conventional biodegradation tests using non-adapted activated sludge, the solvent is generally considered being persistent. The object of the described work was to show, whether and how activated sludge can be adapted to N-methylmorpholine-N-oxide and whether it is possible to purify NMMO-containing wastewaters in conventional wastewater treatment plants. The experiments showed that the sludge can be adapted within about 15–20 days. Adapted sludge can degrade the substance itself and its most important metabolites to concentrations below their detection levels and retain this ability even during limited periods without solvent being present in the wastewater. The main requirement for a successful adaptation is a high sludge age. The degradation takes place in several steps. First, NMMO is reduced to N-methylmorpholine. The next step is a demethylation of N-methylmorpholine to morpholine. This step is crucial for the adaptation process. Once morpholine has been formed, the adaptation proceeds very quickly until none of the substances in question can be detected any longer. So the next step must be the cleavage of the morpholine ring structure.     

Fourier transform infrared spectroscopy as a metabolite fingerprinting tool for monitoring the phenotypic changes in complex bacterial communities capable of degrading phenol

The coking process produces great volumes of wastewater contaminated with pollutants such as cyanides, sulfides and phenolics. Chemical and physical remediation of this wastewater removes the majority of these pollutants; however, these processes do not remove phenol and thiocyanate. The removal of these compounds has been effected during bioremediation with activated sludge containing a complex microbial community. In this investigation we acquired activated sludge from an industrial bioreactor capable of degrading phenol. The sludge was incubated in our laboratory and monitored for its ability to degrade phenol over a 48 h period. Multiple samples were taken across the time‐course and analysed by Fourier transform infrared (FT‐IR) spectroscopy. FT‐IR was used as a whole‐organism fingerprinting approach to monitor biochemical changes in the bacterial cells during the degradation of phenol. We also investigated the ability of the activated sludge to degrade phenol following extended periods (2–131 days) of storage in the absence of phenol. A reduction was observed in the ability of the microbial community to degrade phenol and this was accompanied by a detectable biochemical change in the FT‐IR fingerprint related to cellular phenotype of the microbial community. In the absence of phenol a decrease in thiocyanate vibrations was observed, reflecting the ability of these communities to degrade this substrate. Actively degrading communities showed an additional new band in their FT‐IR spectra that could be attributed to phenol degradation products from the ortho‐ and meta‐cleavage of the aromatic ring. This study demonstrates that FT‐IR spectroscopy when combined with chemometric analysis is a very powerful high throughput screening approach for assessing the metabolic capability of complex microbial communities.     

The utilization of morpholine as a sole nitrogen source by Gram-negative bacteria

The ability of Gram-negative bacteria to degrade morpholine when growing in pure culture is reported for the first time. Several bacterial strains were able to degrade morpholine and to utilize it as a sole nitrogen source but not as a sole carbon and energy source. The organisms studied were obtained from river water and activated sludge and could not be isolated directly on morpholine-containing media which always yielded growth of Gram-positive bacteria using morpholine as a carbon and energy source. The Gram-negative strains were isolated on the basis of their ability to grow on the structurally-related heterocyclc amines piperidine and pyrrolidine.     

Biodegradation of Malathion with Indigenous Acclimated Activated Sludge in Batch Mode and in Continuous-Flow Packed-Bed Reactor

Acclimated activated sludge was examined for its ability to degrade malathion with and without the presence of glucose as a potential cometabolite substrate. In this study, a packed-bed reactor (PBR) using three kinds of biofilm carriers was employed for efficient degradation of malathion. The results obtained indicate that microorganisms tested were able to degrade malathion. The observed degradation rate of the pesticide in the presence of glucose was the same as without glucose. The activated sludge was found to be able to use malathion as the sole phosphorus source. In contrast, the degradation ability of the activated sludge was lost when the pesticide was used as the sole source of sulfur. The degradation capacity of the PBR was higher than the performance obtained with the batch reactor. The reactor packed with crushed olive kernels exhibited the best performance, allowing a total removal of malathion (10 mg/dm³) within 12 h.     

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.     

Instability of the morpholine-degradative phenotype in mycobacteria isolated from activated sludge

The stability of the morpholine-degradative phenotype was studied in a group of nine distinct strains of mycobacteria, all isolated from an activated sludge plant treating morpholine.
Variants incapable of morpholine degradation arose from all strains at high frequency following growth under non-selective conditions. However, strains differed with respect to the frequency at which Mor⁻ variants arose. No spontaneous reversion to wild type could be demonstrated. Six of the nine mycobacterial strains contained plasmids, each had a different plasmid profile. The plasmid profiles of wild type and Mor⁻ variants were compared. In no case could loss of the ability to grow on morpholine be correlated with loss of a complete plasmid. However, evidence is presented which suggests that in two strains loss of the morpholine-degradative phenotype may be correlated with a decrease in the size of a very large plasmid implying deletion of the DNA encoding morpholine degradation. The techniques employed to screen for plasmids in mycobacteria are discussed.     

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.     

Effect of activated carbon on the biological treatment of oil-water emulsions

Waste water, derived from the reprocessing of used emulsions or suspensions, contains high concentrations of emulsified mineral oil and stabilizers, as well as different additives that are needed during the treatment process. Two stirred-tank reactors and two fixed-bed reactors were used to study the biodegradation of these waste-water compounds during two-stage biological treatment. The waste water was first proceesed in an activated sludge reactor to remove easily biodegradable substances. The effluent from the first stage was treated in three parallel operating reactors: an activated sludge tank containing different amounts of powdered activated carbon (PAC, between 0 and 2%), an upflow anaerobic fixed-bed reactor and an aerobic fixed-bed reactor (trickling filter). The results from the continuous treatment were compared with laboratory batch experiments. About 60% of the influent TOC was reduced by the first activated sludge treatment. The removal efficiency increased to about 70% by using a second activated sludge stage. This degradation was comparable to the maximum degree of degradation measured in laboratory batch experiments. PAC addition to the second activated sludge tank resulted in increased degradation rates. The removal efficiency increased to about 76% when 0.1% PAC was added and to 96% with 1% PAC. The removal efficiency decreased to 84% when the proportion of PAC was further increased to 2%. Variations in the amount of PAC addition per unit influent volume in the range of 50 and 200 mg/l had no significant effect on the TOC removal. Degradation models based on the MONOD-type equation were found to be in close correlation with the results obtained from batch experiments. However, the biological removal rates measured in batch experiments did not reflect the removal capacity determined in continuous operating treatment systems.     

Evaluation of the biodegradation potential of 1,4-dioxane in river, soil and activated sludge samples

To evaluate the biodegradation potential of 1,4-dioxane in natural environments, a total of 20 environmental samples including river water, activated sludge, soil from the drainage area of a chemical factory and garden soil were subjected to a 1,4-dioxane degradation test. The five soil samples from the drainage area of the chemical factory were capable of reducing 100 mg l^?1 of 1,4-dioxane to below the detection limit (0.8 mg l^?1) within 33 days. In one activated sludge sample, 100 mg l^?1 of 1,4-dioxane decreased by 69% within 14 days via cometabolic degradation in the presence of 100 mg l^?1 of tetrahydrofuran (THF). The ability of all samples to degrade 1,4-dioxane degradation with or without THF increased after repeated enrichment, except for one soil sample from the drainage area of the chemical factory that was no longer able to degrade 1,4-dioxane after the third cycle of enrichment. However, most of the samples (14/20) were not able to degrade 1,4-dioxane degradation. Thus, it can be concluded that the potential for 1,4-dioxane degradation is not ubiquitously distributed in natural environment.     

The microbial degradation of morpholine

Morpholine can be completely degraded microbiologically, and two organisms have been isolated, each capable of growth in a simple mineral salts medium with morpholine as the sole source of carbon, nitrogen and energy. Excess nitrogen is liberated as ammonia. The enzymes responsible for the oxidation of morpholine are inducible and, in organism Mor G, will also oxidize piperidine, piperazine and pyrrolidine, which are not growth substrates. Ethanolamine is a likely intermediate, though the metabolic steps in morpholine degradation do not give rise solely to acetyl-CoA. After a period of acclimation, a laboratory scale activated sludge plant effectively removed morpholine over the long period it was operated; the sludge was also capable of nitrification. The possible effects of other chemicals in trade wastes containing morpholine on nitrification and morpholine oxidation are described.     

Biodegradation of acetonitrile by adapted biofilm in a membrane-aerated biofilm reactor

A membrane-aerated biofilm reactor (MABR) was developed to degrade acetonitrile (ACN) in aqueous solutions. The reactor was seeded with an adapted activated sludge consortium as the inoculum and operated under step increases in ACN loading rate through increasing ACN concentrations in the influent. Initially, the MABR started at a moderate selection pressure, with a hydraulic retention time of 16 h, a recirculation rate of 8 cm/s and a starting ACN concentration of 250 mg/l to boost the growth of the biofilm mass on the membrane and to avoid its loss by hydraulic washout. The step increase in the influent ACN concentration was implemented once ACN concentration in the effluent showed almost complete removal in each stage. The specific ACN degradation rate achieved the highest at the loading rate of 101.1 mg ACN/g-VSS h (VSS, volatile suspended solids) and then declined with the further increases in the influent ACN concentration, attributed to the substrate inhibition effect. The adapted membrane-aerated biofilm was capable of completely removing ACN at the removal capacity of up to 21.1 g ACN/m² day, and generated negligible amount of suspended sludge in the effluent. Batch incubation experiments also demonstrated that the ACN-degrading biofilm can degrade other organonitriles, such as acrylonitrile and benzonitrile as well. Denaturing gradient gel electrophoresis studies showed that the ACN-degrading biofilms contained a stable microbial population with a low diversity of sequence of community 16S rRNA gene fragments. Specific oxygen utilization rates were found to increase with the increases in the biofilm thickness, suggesting that the biofilm formation process can enhance the metabolic degradation efficiency towards ACN in the MABR. The study contributes to a better understanding in microbial adaptation in a MABR for biodegradation of ACN. It also highlights the potential benefits in using MABRs for biodegradation of organonitrile contaminants in industrial wastewater.     

Biological uptake of influent organic matter as an electron donor for denitrification by activated sludge

To preserve organic materials in the sludge required for denitrification, a study was made with a contact process. The contactor, when operated with a short retention time (30 min), gave incomplete metabolism of organic matter. With 64% of the influent soluble COD (SCOD) was adsorbed to activated sludge within 30 min. The specific mass of organic matter uptaken was 55 mg SCOD/g Mixed Liquor Suspended Solids that enhanced the denitrification efficiency up to 63% in the following denitrification step.     

Microbial degradation of nitrobenzene and mono-nitrophenol by bacteria enriched from municipal activated sludge.

Using a mixture of three mono nitrophenols as sole carbon, nitrogen and energy sources, mixed cultures were enriched from municipal activated sludge to degrade both nitrophenols and nitrobenzene. Bacterial growth and degradation rate could be increased by supplementing the medium with 0.1% YE. Microorganisms were isolated from the nitrophenols enrichment, and they were identified as strains of Comamonas testosteroni and Acidovorax delafieldii. These strains showed broad degradation ability toward nitrophenols and nitrobenzene.     

The effect of pH on the efficiency of an SBR processing piggery wastewater

To treat piggery wastewater efficiently, the hydrolysis of urea (mainly derived from swine urine) in piggery wastewater with the change of sewage pH must be considered. Using activated sludge, piggery wastewater was treated in a sequencing batch reactor (SBR), and the effects of influent pH on SBR processing efficiency, sludge settle ability, and sludge activity were investigated. The results showed that a high influent pH value contributed to the improvement of the removal rate of ammonia nitrogen and reduction of the chemical oxygen demand (COD). When the influent pH was between 9.0 and 9.5, the removal rate of ammonia nitrogen was higher than 90%, and the reduction of COD from its original value was 80%. The influent pH had a greater influence on sludge concentration and sludge activity. When the influent pH increased from 7.0 to 9.5, the sludge concentration increased from 2,350 to 3,947 mg/L in the reactor, and the activities of ammonium oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) first increased and then decreased. When the influent pH was 9.0 and 8.0, the maximum values (0.48 g O₂/(g MLSS/day) and 0.080 g O₂/(g MLSS/day)) were reached, and the sludge settling ratio was nearly steady between 20 and 35% in each reactor.     

Effect of hexavalent chromium on the activated sludge process and on the sludge protozoan community

The objectives of this study were the determination of chromium effects to the performance of an activated sludge unit and the investigation of the response of the activated sludge protozoan community to Cr(VI). Two bench scale activated sludge reactors were supplied with synthetic sewage containing Cr(VI), at concentrations from 1 up to 50 mg L(-1). Protozoan species were identified and were related to the system efficiency. Variations in the abundance and diversity of the protozoan species were observed under various chromium concentrations. High removal rates of organics and nutrients were observed after the acclimatization of the activated sludge, which were related to the initial chromium(VI) concentration. Chromium(VI) removal efficiency was high in all cases. The protistan community was affected by the influent chromium content. Dominance of sessile species was observed in the reactor receiving 5 mg L(-1) influent chromium, whereas co-dominance of sessile and carnivorous species was observed in the reactors receiving higher chromium concentrations.     

Evaluation of methods to solubilize and analyze cell-associated ectoenzymes

A protocol for production, storage, and use of Shock 1 (Shk1) bioreporter cells for toxicity monitoring in wastewater treatment facilities was developed. Shk1 is a bioluminescent toxicity bioreporter for activated sludge previously constructed by the incorporation of lux genes into an activated sludge microorganism.

A number of factors affecting Shk1 growth and bioluminescence were examined including the growth medium, tetracycline concentration, storage conditions, and test media. Based on the results of these experiments, a toxicity testing protocol was developed that involved growth of cultures in nutrient broth with tetracycline, storage of cultures at 4 °C, cell activation by reinoculation into nutrient broth, and toxicity testing by cell injection into the test media. Effective use of this approach required standardized time intervals for cell growth, storage, activation and exposure in the test media.

Bioluminescence from Shk1 cells was measured in nutrient broth and influent wastewater and activated sludge mixed liquor from a municipal wastewater treatment plant. Using the Shk1 toxicity testing protocol, Zn EC₅₀ values for bioluminescence in nutrient broth, influent wastewater, and activated sludge mixed liquor were approximately 42, 7, and 32 mg/l, respectively. Zn concentrations as low as 1 mg/l could be detected in influent wastewater. The detection limit in influent wastewater is below the Zn concentrations typically reported to affect the activated sludge process.     

Biodegradation of naphthalene in the oil refinery wastewater by enriched activated sludge

The main purpose of this paper is to study naphthalene (NAP) biodegradation by acclimated activated sludge, employing the culture-enrichment method in a continuous flow bioreactor of the wastewater treatment process. The effects of various COD loadings and influent flow rates of an artificial wastewater containing 15 mg l⁻¹ NAP on the biodegradation rates of the activated sludge will be investigated, in order to determine the biodegradation kinetics and minimum mean cell residence time of the activated sludge. From the experimental results, it was found that the resulting enriched activated sludge follows the growth rate of the Monod type and can biodegrade those COD and NAP loadings in the influents efficiently, and its bio-treatment efficiency on NAPs increases with the decrease of influent flow rate. The sludge volume index (SVI) of the resulting enriched activated sludge meets the design value required by the convectional activated sludge process for the treatment of wastewater.     

一株氯霉素降解细菌的分离鉴定与代谢特性研究

微生物是介导环境中氯霉素降解转化的主要驱动者,但高效降解矿化菌株资源匮乏,氧化反应介导的代谢途径不清。为研究微生物介导下氯霉素的环境归趋过程,为氯霉素污染环境强化修复提供菌株资源,文中以受氯霉素污染的活性污泥为接种源,首先富集获得一个由红球菌Rhodococcus主导 (相对丰度>70%) 的氯霉素高效降解菌群,并从中分离获得一株能够高效降解氯霉素的菌株CAP-2,通过16S rRNA基因分析鉴定为红球菌Rhodococcus sp.。菌株CAP-2能在不同营养条件下高效降解氯霉素。基于菌株CAP-2对检测到的代谢产物对硝基苯甲酸和已报道的代谢产物对硝基苯甲醛和原儿茶酸的生物转化特征,提出其降解途径是由氯霉素侧链氧化断裂生成对硝基苯甲醛,进一步氧化为对硝基苯甲酸的新型氧化降解途径。该菌株对于氯霉素分解代谢的分子机制研究以及受氯霉素污染环境的原位生物修复应用具有巨大潜力。     

Degradation of Morpholine by an Environmental Mycobacterium Strain Involves a Cytochrome P-450

A Mycobacterium strain (RP1) was isolated from a contaminated activated sludge collected in a wastewater treatment unit of a chemical plant. It was capable of utilizing morpholine and other heterocyclic compounds, such as pyrrolidine and piperidine, as the sole source of carbon, nitrogen, and energy. The use of in situ ¹H nuclear magnetic resonance (¹H NMR) spectroscopy allowed the determination of two intermediates in the biodegradative pathway, 2-(2-aminoethoxy)acetate and glycolate. The inhibitory effects of metyrapone on the degradative abilities of strain RP1 indicated the involvement of a cytochrome P-450 in the biodegradation of morpholine. This observation was confirmed by spectrophotometric analysis and ¹H NMR. Reduced cell extracts from morpholine-grown cultures, but not succinate-grown cultures, gave rise to a carbon monoxide difference spectrum with a peak near 450 nm, which indicated the presence of a soluble cytochrome P-450. ¹H NMR allowed the direct analysis of the incubation medium containing metyrapone, a specific inhibitor of cytochrome P-450. The inhibition of morpholine degradation was dependent on the morpholine/metyrapone ratio. The heme-containing monooxygenase was also detected in pyrrolidine- and piperidine-grown cultures. The abilities of different compounds to support strain growth or the induction of a soluble cytochrome P-450 were assayed. The results suggest that this enzyme catalyzes the cleavage of the C—N bond of the morpholine ring.     

Preliminary studies on continuous chromium(VI) biological removal from wastewater by anaerobic-aerobic activated sludge process

The long-term continuous chromium(VI) removal from synthetic wastewater affected by influent hexavalent chromium (Cr(VI)) and glucose concentrations were studied with an anaerobic-aerobic activated sludge process. It was observed that before activated sludge was acclimated, the chromium in the effluent increased immediately as the influent chromium increased. However, both Cr(VI) and total chromium (TCr) in the effluent significantly decreased after acclimation. In the acclimated activated sludge, the chromium removal efficiency was 100% Cr(VI) and 98.56% TCr at influent Cr(VI) levels of 20 mg/day, 100% Cr(VI) and 98.92% TCr at influent Cr(VI) levels of 40 mg/day, and 98.64% Cr(VI) and 97.16% TCr at influent Cr(VI) levels of 60 mg/day. The corresponding effluent Cr(VI) and TCr concentrations were 0 and 0.012 mg/l, 0 and 0.018 mg/l, and 0.034 mg/l and 0.071 mg/l, respectively. When the influent glucose increased from 1125 to 1500 mg/l at influent Cr(VI) dosage of 60 mg/day, the Cr(VI) and TCr removal efficiency with the acclimated activated sludge improved from 98.64% and 97.16% to 100% and 98.48%, respectively, and the chromium concentration in the effluent decreased from 0.034 mg/l of Cr(VI) and 0.071 mg/l of TCr to 0 (Cr(VI)) and 0.038 mg/l (TCr). The effluent COD and turbidity was around 40 mg/l and 0, respectively, after the activated sludge was acclimated. Further studies showed that after the activated sludge was acclimated, its specific dehydrogenases activity (SDA) and protein contents increased. The SDA and protein increased respectively 15% and 10% when influent Cr(VI) increased from 20 to 60 mg/day.