Optimisation of process parameters for bioreduction of azo dyes using Bacillus firmus under batch anaerobic condition

A new dye decolourising bacterial strain was isolated from textile wastewater and identified as Bacillus firmus. The study indicated that the bacterium could efficiently decolourise different azo dyes under static culture conditions. Characterisation of the efficiency of azo dye reduction by this isolate using both spectral and HPLC analysis was found to be a function of process parameters which include dye concentration, culture broth pH, incubation temperature, aeration as well as nitrogen source. For decolourisation, the optimal pH and temperature were 7–8 and 20–35°C respectively, while remarkable dye degradation was obtained within 18 h for dye concentrations below 100 mg L^?1. With the addition of yeast extract and under optimal conditions, dye reduction was enhanced and complete colour removal was achieved within 12 h. Colour removal was shown to be due to biodegradation rather than adsorption of dyes on bacterial cells. This study confirms the ability of the new dye‐degrading strain, Bacillus firmus, to decolourise and degrade different azo dyes and highlights its high biotechnology potential for the eco‐friendly treatment of textile wastewater when optimal conditions are applied.     

Aerobic degradation of acid orange 7 in a vertical-flow constructed wetland

Biological, aerobic degradation of an azo dye and of the resultant, recalcitrant, aromatic amines in a constructed wetland (CW) was demonstrated for the first time. A vertical-flow CW, planted with Phragmites sp. was fed with 127 mg l⁻¹ of acid orange 7 (AO7) at hydraulic loads of 28, 40, 53 and 108 l m⁻²day⁻¹. Color removal efficiencies of up to 99% clearly demonstrate cleavage of the azo bond, also confirmed by the similar AO7 removal and SO₄²⁻ release rates revealing that adsorption onto the matrix was constant. The positive redox potential at the outlet demonstrates that aerobic conditions were present. Chemical oxygen demand and total organic carbon removal efficiencies of up to 93% were also indicative of AO7 mineralization. The degradation of sulfanilic acid was confirmed by the presence of NO₃⁻, SO₄²⁻ and secondary metabolites, which suggest at least two degradation pathways leading to a common compound, 3-oxoadipate.     

Biodegradation potential of pure and mixed bacterial cultures for removal of 4-nitroaniline from textile dye wastewater

Environmentally toxic aromatic amines including nitroanilines are commonly generated in dye contaminated wastewater in which azo dyes undergo degradation under anaerobic conditions. The aim of this study was to develop a process for biological treatment of 4-nitroaniline. Three bacteria identified as Acinetobacter sp., Citrobacter freundii and Klebsiella oxytoca were isolated from enrichment cultures of activated sludge on 4-nitroaniline, after which the isolates and the mixed culture were studied to determine optimal conditions for biodegradation. HPLC analyses showed the mixed culture was capable of complete removal of 100 μmol/L of 4-nitroaniline within 72 h under aerobic conditions. There was an inverse linear relationship (R² = 0.96) between the rate of degradation (V) and 4-nitraoaniline concentrations [S] over 100-1000 μmol/L. The bacterial culture was also capable of decolorizing structurally different azo dyes (Acid Red-88, Reactive Black-5, Direct Red-81, and Disperse Orange-3) and also degraded nitrobenzene. Our findings show that enrichment cultures from activated sludge can be effective for the removal of dyes and their toxic intermediates, and that treatment may best be accomplished using an anaerobic-aerobic process.     

Characterization of degradation process of cyanobacterial hepatotoxins by a gram-negative aerobic bacterium

A bacterium termed 7CY, capable of decomposing cyanobacterial toxins, was isolated from surface water sample of Lake Suwa and degradation of microcystin-RR and nodularin-Har was investigated. The isolated 7CY was a gram-negative, aerobic bacillus, and a member of a genus Sphingomonas. The strain degraded microcystin-LY, -LW, and -LF completely as well as microcystin-LR within 4 days after their addition (6 microg/ml) whereas degradation of nodularin-Har did not occur at all during experiment. On the contrary, the strain was capable of degrading nodularin-Har in the presence of microcystin-RR and both toxins were completely decomposed within 6 days. The strain scarcely degraded nodularin-Har in the presence of microcystin-RR when glucose and ammonium chloride were added to the medium. The degradation of nodularin-Har did not occur in the medium from which bacterial cells had been removed after degradation of microcystin-RR. Furthermore, when microcystin-RR and nodularin-Har were added to the cytoplasm fraction of 7CY cells, microcystin-RR was rapidly degraded within 18 h, but nodularin-Har was not. The strain 7CY may require an enzyme(s) induced during the degradation of microcystin-RR in order to utilize nodularin-Har as nutrition.     

Biodegradation and detoxification potential of rotating biological contactor (RBC) with Irpex lacteus for remediation of dye-containing wastewater

Use of fungal organisms in rotating biological contactors (RBC) for bioremediation of liquid industrial wastes has so far been limited in spite of their significant biodegradation potential. The purpose was to investigate the power of RBC using Irpex lacteus for decolorization and detoxification of industrial dyes and dyeing textile liquors. Recalcitrant dye Methylene Blue (150 mg L⁻¹) was decolorized within 70 days, its mutagenicity removed, and the biological toxicity decreased more than 10-fold. I. lacteus biofilm in the RBC completely decolorized within 26 and 47 days dyeing liquors containing disperse or reactive dyes adjusted to pH4.5 and 5-fold diluted with the growth medium, respectively. Their respective biological toxicity values were reduced 10- to 10⁴-fold in dependence of the test used. A battery of toxicity tests comprising Vibrio fisheri, Lemna minor and Sinapis alba was efficient to monitor the toxicity of textile dyes and wastewaters. Strong decolorization and detoxification power of RBC using I. lacteus biofilms was demonstrated.     

Combined anaerobic-aerobic treatment of azo dyes--a short review of bioreactor studies

The most logical concept for the removal of azo dyes in biological wastewater treatment systems is based on anaerobic treatment, for the reductive cleavage of the dyes' azo linkages, in combination with aerobic treatment, for the degradation of the products from azo dye cleavage, aromatic amines. Since the 1990s, several research papers have been published on combined, sequential or integrated, anaerobic-aerobic bioreactor treatment of azo dye-containing wastewater. The extent of azo dye reduction in the anaerobic phase of those bioreactor systems was generally high, albeit the process often required long reaction times, a limitation that can easily be remedied by making use of the property of redox mediators to speed up the process. The consequent removal of aromatic amines under aerobic conditions was less unequivocal. Although analytical data indicate that many of the aromatic amines were removed from the wastewater, and although the limited amount of available toxicity data all show far-reaching detoxification during aerobic treatment, it is clear that not all aromatic amines can be completely mineralized.     

Carbon cycling in a zero-discharge mariculture system

Interest in mariculture systems will rise in the near future due to the decreased ability of the ocean to supply the increasing demand for seafood. We present a trace study using stable carbon and nitrogen isotopes and chemical profiles of a zero-discharge mariculture system stocked with the gilthead seabream (Sparus aurata). Water quality maintenance in the system is based on two biofiltration steps. Firstly, an aerobic treatment step comprising a trickling filter in which ammonia is oxidized to nitrate. Secondly, an anaerobic step comprised of a digestion basin and a fluidized bed reactor where excess organic matter and nitrate are removed. Dissolved inorganic carbon and alkalinity values were higher in the anaerobic loop than in the aerobic loop, in agreement with the main biological processes taking place in the two treatment steps. The δ¹³C of the dissolved inorganic carbon (δ¹³C_DIC) was depleted in ¹³C in the anaerobic loop as compared to the aerobic loop by 2.5-3‰. This is in agreement with the higher dissolved inorganic carbon concentrations in the anaerobic loop and the low water retention time and the chemolithotrophic activity of the aerobic loop. The δ¹³C and δ¹⁵N of organic matter in the mariculture system indicated that fish fed solely on feed pellets. Compared to feed pellets and particulate organic matter, the sludge in the digestion basin was enriched in ¹⁵N while δ¹³C was not significantly different. This latter finding points to an intensive microbial degradation of the organic matter taking place in the anaerobic treatment step of the system.     

Improvement of RNA-SIP by pyrosequencing to identify putative 4-n-nonylphenol degraders in activated sludge

Nonylphenols (NP) have estrogenic potential because of their phenolic ring, but the organisms involved in the degradation of this alkylated phenol remain unidentified. Using 16S ribosomal RNA (rRNA)-based stable isotope probing (SIP) and a new method based on pyrosequencing, we identified the bacteria involved in the degradation of the aromatic ring of [U-ring-¹³C] 4-n-NP in aerobic sludge. The first order degradation rate of 4-n-NP was 5.5 d⁻¹. Single strand conformation polymorphism of density-separated labeled and unlabeled 16S rRNA showed significant differences and enabled selection of four representative fractions for pyrosequencing. Nineteen phylotypes showed a significant enrichment in the heavy fraction in the labeled pulse. The relative abundances of these phylotypes were combined with the RNA concentration of each fraction to yield a simple model of the distribution of each phylotype across the gradient. This model was used to estimate the percentage of labeling for each phylotype. The sequences showing the highest labeling (11%) were closely related to Afipia sp. but represented only 2 % of the RNA in the heavy fraction of the labeled pulse. The sequences representing the largest proportion of the RNA in the heavy fraction were related to Propionibacterium acnes and Frateuria aurantia, which are known to possess enzymes for phenol degradation. The model shows that despite Afipia having the highest ¹³C enrichment, other species encoding phenol degradation pathways are responsible for more ¹³C incorporation. Last, we showed that some species represent 12% of the total RNA but contain only 1% ¹³C above natural abundance.     

Biodegradability of kraft mill TCF biobleaching effluents: Application of enzymatic laccase-mediator system

The great amount of pollutants released from kraft pulp processes, mainly from cooking and bleaching stages, is one of the most relevant environmental problems in this type of industry. New bleaching sequences are being studied based on the use of oxidative enzymes from fungal cultures. In this study, the bleaching systems consisting of Laccase and different mediators such as 1-hydroxybenzotriazole, violuric acid, syringaldehyde and methyl syringate in the bleaching sequence of Eucalyptus globulus kraft pulp were applied. The main objective of this study is to evaluate the aerobic and anaerobic biodegradability and toxicity to Vibrium fischeri of generated L-stage and total bleaching sequence effluents.The highest levels of aerobic and anaerobic degradation of the generated effluents were achieved for treatments with laccase plus violuric acid, with 80% of aerobic degradation and 68% of anaerobic biodegradation. V. fischeri toxicity was remarkably reduced for all the effluents after aerobic degradation.     

Some effects of pulp and paper wastewater on microbiological water quality of a river

A study was conducted to detect the source of fecal indicator bacteria and measure the resulting impairment of water quality of the Sturgeon River at Sturgeon Falls, Ontario, Canada. The bacteriological water quality of the river above the dam at Sturgeon Falls was fairly good, while below the dam the water quality was degraded. The principal input of fecal indicator bacteria was traced to the paper mill at Sturgeon Falls. The impairment of water quality was detected all the way to recreational areas near the mouth of the river. The impaired stretch of the river had diminished aesthetic appeal. Other sources of indicator bacteria could be masked by the large numbers of similar bacteria discharged from the paper mill. The principal fecal coliform was Klebsiella pneumoniae. In pulp mill wastes the fecal origin of this bacterium can be disputed, and so its presence in large numbers interfered with the interpretation of fecal coliform results. K. pneumoniae is also an opportunistic pathogen which causes infections in humans. Thermotolerant oxidase positive bacteria which were isolated from the paper mill wastes, register as false positives in the fecal coliform test causing problems in interpretation. Pseudomonas aeruginosa was detected in the pulp mill wastewater, and at a level of 82 PA 100 ml⁻¹ in recreational areas of the river. This was considered to be of some hazard to users of the water. In addition, Escherichia coli was detected in the mill wastewater, at about 200 EC 100 ml⁻¹, and this probably indicated some measure of fecal pollution though likely of animal origin. Finally the nutrient rich wastewater led to an approx. 50-fold increase in density of aerobic heterotrophic bacteria in the river water, as well as the production of a slimy filamentous growth on surfaces, stones and wooden pilings, in the river. The principal organism in this slime was the fungus Leptomitus.     

Degradation of phthalate esters in an activated sludge wastewater treatment plant

Efficient removal of phthalate esters (PE) in wastewater treatment plants (WWTP) is becoming an increasing priority in many countries. In this study, we examined the fate of dimethyl phthalate (DMP), dibutyl phthalate (DBP), butylbenzyl phthalate (BBP), and di-(2-ethylhexyl) phthalate (DEHP) in a full scale activated sludge WWTP with biological removal of nitrogen and phosphorus. The mean concentrations of DMP, DBP, BBP, and DEHP at the WWTP inlet were 1.9, 20.5, 37.9, and 71.9 μg/L, respectively. Less than 0.1%, 42%, 35%, and 96% of DMP, DBP, BBP, and DEHP was associated with suspended solids, respectively. The overall microbial degradation of DMP, DBP, BBP, and DEHP in the WWTP was estimated to be 93%, 91%, 90%, and 81%, respectively. Seven to nine percent of the incoming PE were recovered in the WWTP effluent. Factors affecting microbial degradation of DEHP in activated sludge were studied using [U-¹⁴C-ring] DEHP as tracer. First order rate coefficients for aerobic DEHP degradation were 1.0×10⁻², 1.4×10⁻², and 1.3×10⁻³ at 20, 32, and 43 °C, respectively. Aerobic degradation rates decreased dramatically under aerobic thermophilic conditions (<0.1×10⁻² h⁻¹ at 60 °C). The degradation rate under anoxic denitrifying conditions was 0.3×10⁻² h⁻¹, whereas the rate under alternating conditions (aerobic-anoxic) was 0.8×10⁻² h⁻¹. Aerobic DEHP degradation in activated sludge samples was stimulated 5-9 times by addition of a phthalate degrading bacterium. The phthalate degrading bacterium was isolated from activated sludge, and maintained a capacity for DEHP degradation while growing on vegetable oil. Collectively, the results of the study identified several controls of microbial PE degradation in activated sludge. These controls may be considered to enhance PE degradation in activated sludge WWTP with biological removal of nitrogen and phosphorus.     

Toxicological effect of indole and its azo dye derivatives on some microorganisms under aerobic conditions

Azo dyes are ubiquitous commercial chemicals that present unique environmental problems. The azo dyes in particular can undergo natural anaerobic degradation to potentially carcinogenic amines. They pose a major problem for water-treatment plants downstream. One strategy to remidate polluted surface contaminants is to make use of the degradative capacity of bacteria rather than using destructive chemical reactions. Therefore, pathogenic and non-pathogenic microorganisms namely; Bacillus thuringiensis, Bacillus subtilis, Bacillus megaterium, Proteus vulgaris, Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli and Saccharomyces cerevisiae have been chosen and the toxicological effect of indole and its azo dye methyl derivatives on these microorganisms was studied under aerobic conditions. While these compounds have showed remarkable activity against B. megaterium, B. subtilis, B. thuringiensis and S. aureus, they did not exhibit any activity against P. vulgaris. However, indole acted as an inhibitor on all of these compounds specially the Gram negative bacterium P. aeruginosa.     

Aerobic biotransformation potential of a commercial mixture of naphthenic acids

The biotransformation potential of a commercial naphthenic acid (NA) mixture (NA sodium salt; TCI Chemicals) under aerobic conditions was investigated using mixed aerobic cultures developed under various levels and duration of NA exposure. A culture enriched using the commercial NA mixture as the sole carbon source degraded NAs in a range of NA concentrations, regardless of culture age and the presence of a co-substrate; however, only 28.5% of the NA-carbon was detected as CO₂ while 44% was utilized for biomass growth. A fraction of the NA mixture (15–26%) was persistent under all conditions studied. In contrast, a culture fed with a degradable synthetic wastewater only (NA un-amended culture) and another culture fed with the same wastewater and 50 mg NA/L (NA-amended culture), over time lost their ability to degrade NAs. Analysis of the 16S rRNA gene based clone library revealed that 80% of the NA-enriched culture belonged to the γ-Proteobacteria class and was largely dominated by phylotypes most closely related to known NA and hydrocarbon degraders such as Pseudomonas and Microbulbifer. The results of this study indicate that although significant NA degradation is possible, only a small fraction of the NA mixture is completely mineralized to CO₂. Further investigation into the biotransformation products and conditions affecting NA biodegradation under realistic refinery and environmental conditions will help to design effective treatment and bioremediation processes.     

Environmental occurrence and degradation of the herbicide n-chloridazon

A sampling campaign was carried out for n-chloridazon (n-CLZ) and its degradation product desphenyl-chloridazon (DPC) in the Hesse region (Germany) during the year 2007: a total of 548 environmental samples including groundwater, surface water and wastewater treatment plant (WWTP) effluent were analysed. Furthermore, aerobic degradation of n-CLZ has been studied utilising a fixed bed bioreactor (FBBR).In surface water, n-CLZ was detected at low concentrations (average 0.01 ± 0.06 μg L⁻¹; maximum 0.89 μg L⁻¹) with a seasonal peak, whereas DPC was present throughout the year at much higher concentrations (average 0.72 ± 0.81 μg L⁻¹; maximum 7.4 μg L⁻¹). Higher n-CLZ concentrations were observed in the North compared with South Hesse, which is ascribed to a higher density of agricultural areas. Furthermore, methylated DPC (Me-DPC), another degradation product, was detected in surface water.In the degradation test, n-CLZ was completely converted to DPC at all concentrations tested (Me-DPC was not formed under the test conditions). DPC was resistant to further degradation during the whole experimental period of 98 days. The results obtained suggest persistence and high dispersion of DPC in the aquatic environment.     

Isolation of Yersinia from surface water: Comparative study of three selective media

Isolation of Yersinia was undertaken on three media from surface water of the Moselle River. Samples collected over an 8-month period were examined with Y-M agar, CAL agar and “Y” medium. Out of 48 water samples, 33.3% were positive. The isolation frequency with Y-M agar (27%) was significantly higher than with CAL agar of “Y” medium (χ² = 11.2; 0.001 < P < 0.01). Furthermore, Y-M agar exhibited a wide diversity of species; Y. enterocolitica, Y. frederiksenii, Y. kristensenii and Y. intermedia were isolated. In addition, the aerobic cold-enrichment technique without culture broth appears adequate for Yersinia isolation.     

Influence of Asellus aquaticus on Escherichia coli, Klebsiella pneumoniae, Campylobacter jejuni and naturally occurring heterotrophic bacteria in drinking water

Water lice, Asellus aquaticus (isopoda), frequently occur in drinking water distribution systems where they are a nuisance to consumers and water utilities. Whether they are solely an aesthetic problem or also affect the microbial water quality is a matter of interest. We studied the influence of A. aquaticus on microbial water quality in non-chlorinated drinking water in controlled laboratory experiments. Pure cultures of the indicator organisms Escherichia coli and Klebsiella pneumoniae and the pathogen Campylobacter jejuni as well as naturally occurring heterotrophic drinking water bacteria (measured as heterotrophic plate counts, HPC) were investigated in microcosms at 7 °C, containing non-sterilised drinking water, drinking water sediment and A. aquaticus collected from a non-chlorinated ground water based drinking water supply system. Concentrations of E. coli, K. pneumoniae and C. jejuni decreased over time, following a first order decay with half lives of 5.3, 18.4 and 1.3 days, respectively. A. aquaticus did not affect survival of indicators and pathogens substantially whereas HPC were influenced by presence of dead A. aquaticus. Growth rates increased with an average of 48% for bacteria grown on R-2A agar and an average of 83% for bacteria grown on yeast extract agar when dead A. aquaticus were present compared to no and living A. aquaticus present. A. aquaticus associated E. coli, K. pneumoniae and C. jejuni were measured (up to 25 per living and 500 per dead A. aquaticus) and so were A. aquaticus associated heterotrophic bacteria (>1.8*10⁴ CFU per living and >6*10⁴ CFU per dead A. aquaticus). A. aquaticus did not serve as an optimised habitat that increased survival of indicators and pathogens, since A. aquaticus associated E. coli, K. pneumoniae and C. jejuni were only measured as long as the bacteria were also present in the water and sediment.     

Biodegradation of triclosan by a wastewater microorganism

Triclosan, a synthetic antimicrobial agent, has been considered as an emerging environmental contaminant. Here we reported a triclosan-degrading wastewater bacterial isolate, Sphingopyxis strain KCY1, capable of dechlorinating triclosan with a stoichiometric release of chloride. The stain can degrade diphenyl ether but not 2,4,4′-tribromodiphenyl ether and 2,2′,4,4′-tetrabromodiphenyl ether, despite all these three compounds are structurally similar to triclosan. While strain KCY1 was unable to grow on triclosan and catechol, it could grow with glucose, sodium succinate, sodium acetate, and phenol. When grown with complex nutrient medium containing a trace amount of triclosan (as low as 5 μg/L), the strain could retain its degradation ability toward triclosan. The maximum-specific triclosan degradation rate (q_m) and the half-velocity constant (K_m) are 0.13 mg-triclosan/mg-protein/day and 2.8 mg-triclosan/L, respectively. As triclosan degradation progressed, five metabolites were identified and these metabolites continue to transform into non-chlorinated end products, which was supported by a sharp drop in androgenic potential. The activity of catechol 2,3-dioxygenase in the cell extract was detected. No triclosan degradation was observed in the presence of 3-fluorocatechol, an inhibitor of meta-cleavage enzyme, suggesting that triclosan degradation proceed via meta-cleavage pathway. Based on all the observations, a degradation pathway for triclosan by strain KCY1 was proposed.     

Natural and acquired resistance of mice to infection by airborne Klebsiella pneumoniae after subchronic intoxication by cadmium administered orally

Mice daily ingested about 22 mg of cadmium per kg of body weight in drinking water for 30 days. On the 30th day, the liver and kidneys of the mice contained about 18 μg of Cd²⁺ perg of fresh organ. A group of these mice was immunized against Klebsiella pneumoniae using two injections of vaccine, the first on the 7th day and the second on the 14th day of intoxication. On the 28th day, the non-immunized and the immunized mice were infected via a respiratory route by one lethal dose 50% of K. pneumoniae (the LD₅₀ for the immunized mice was 2.4 times higher than the LD₅₀ for the non-immunized mice). Comparison with the non-intoxicated control mice showed that the ingestion of Cd²⁺ did not significantly modify the natural resistance or the acquired resistance of the mice to the infection by airborne K. pneumoniae.     

Evaluation of natural and enhanced PCP biodegradation at a former pesticide manufacturing plant

Pentachlorophenol (PCP) has been used in the past as a pesticide, herbicide, antifungal agent, bactericide, and wood preservative. Thus, PCP is among the most ubiquitous chlorinated compounds found in groundwater contamination. A former pesticide manufacturing plant located in southern Taiwan has been identified as a PCP spill site. In this study, groundwater samples collected from the PCP site were analyzed to assess the occurrence of natural PCP biodegradation. Microcosm experiments were conducted to (1) evaluate the feasibility of biodegrading PCP by indigenous microbial consortia under aerobic and cometabolic conditions, and (2) determine the potential of enhancing PCP biodegradation using cane molasses and biological sludge cake as the substitute primary substrates under cometabolic conditions. The inocula used in this microcosm study were aquifer sediments collected from the PCP site and activated sludges collected from the municipal and industrial wastewater treatment plants. Results from this field investigation indicate that the natural biodegradation of PCP is occurring and causing the decrease in PCP concentration. Microcosm results show that the indigenous microorganisms can biodegrade PCP under both aerobic and aerobic cometabolism conditions. A PCP-degrading bacterium was isolated from the collected aquifer sediments and identified as Pseudomonas mendocina NSYSU via some biochemical tests and further conformation of DNA sequencing. In batch cultures, P. mendocina NSYSU used PCP as its sole source of carbon and energy. The isolated bacterium, P. mendocina NSYSU, was capable of completely degrading PCP as indicated by the increase in biomass formation with the decrease in PCP concentrations occurred in the carbon-free medium simultaneously. Results indicate that the in situ or on-site aerobic bioremediation using indigenous microorganisms or inoculated bacteria would be a feasible technology to clean up the studied PCP-contaminated site. Results from this study will be useful in designing a scale-up in situ or on-site PCP bioremediation system (e.g., on-site bioreactor) for field application.     

A new interpretation of endogenous respiration profiles for the evaluation of the endogenous decay rate of heterotrophic biomass in activated sludge

In current activated sludge models aerobic degradation, resulting in loss of activity and mass of activated sludge is expressed with only one process called decay. The kinetics of this process is regarded to be first order and constant with respect to the loading conditions. In this work twelve aerobic digestion batch experiments were conducted for the activated sludge of seven different water resource recovery facilities (WRRFs). An analysis of the obtained respirograms shows three clearly distinguishable phases. The first phase is assumed to be due to the degradation of stored material (X_STOR) and active biomass simultaneously. The second phase is exclusively due to the degradation of active biomass that is regarded to consist mainly of ordinary heterotrophic biomass (X_OHO). The first order decay rate is slower than the degradation rate in phase 1 and varies between samples. The decay rate correlates with the activity of the activated sludge expressed as the ratio of initial heterotrophic OUR and the initial organic fraction X_ORG of the activated sludge. This second phase was detectable until day 5 of most of the experiments. After that time within phase 3 the OUR decrease slows down and the OUR even increased for short intervals. This behaviour is thought to be due to the activity of higher organisms and the adaptation of microorganisms to starvation.