Studies on Induction and Repression in Activated Sludge Systems

Both repression and induction of substrate utilization have been the subject of many basic research investigations employing pure cultures. In this investigation these effects were studied using heterogeneous microbial populations prevalent in such biological treatment processes as activated sludge systems.Diauxic substrate removal by activated sludge was observed in a multicomponent medium consisting of glucose and sorbitol. The sludge was acclimated solely to sorbitol; however, the presence of glucose blocked sorbitol removal until glucose was completely utilized. Both diphasic and triphasic oxygen utilization was shown for activated sludges metabolizing multicomponent synthetic wastes consisting of glucose, melibiose, and lactose. It appears from these studies that melibiose utilization was suppressed by the presence of glucose and, although melibiose induced acclimation to lactose, the presence of melibose suppressed lactose utilization. Studies were also conducted using glycogen and starch systems in which it was found that acclimation to either compound conferred immediate acclimation to the other. It was also found that loss of acclimation to lactose was a passive phenomenon and its kinetics could be predicted on the basis of simple diluting out of the enzyme(s) responsible for such acclimation.     

Aerobic Heterotrophic Bacterial Populations of Sewage and Activated Sludge: III. Adaptation in a Synthetic Waste

The nature and behavior of the microbial population developed on a synthetic waste containing salts and sorbitol are compared to that of a treatment-plant activated sludge. The population of the adapted sludge developed on the synthetic waste consisted approximately of only six kinds of bacteria. Experiments with radioactive sorbitol indicate that the microbial population developed on the synthetic waste showed the effects of repression by glucose on the metabolism of sorbitol; in contrast, the activated sludge from a plant treating primarily domestic waste was able to attack both substrates immediately and simultaneously.     

Gluco-oligosaccharide synthesis by free and immobilized beta-glucosidase

Gluco-oligosaccharides were synthesized through the enzymatic condensation of D-glucose at high concentration using a commercial almond beta-glucosidase. The synthesis reactions were carried out with both free and immobilized enzyme, with or without sorbitol, an efficient depressor of water activity (a(w)) in the presence of different glucose concentrations. The yield and the composition of the gluco-oligosaccharides produced changed with the reaction mixture and the form of the enzyme used (free or immobilized). The use of 5 M glucose solution permitted only disaccharides to be obtained, whereas with a glucose concentration of 7.5 M glucose, di-, tri-, and tetrasaccharides were produced. A 7.5 M glucose solution used with 4.4 M sorbitol gave three times more disaccharides than the same solution without sorbitol. Moreover, the immobilized enzyme was much more active in synthesis. The synthesis yield (oligomers mg/mL . mg of enzyme) after immobilization was 573% compared to that of the free enzyme, when a 7.5 M glucose solution was tested. The effects of substrate concentration, sorbitol addition and enzyme immobilization were investigated. (c) 1993 John Wiley & Sons, Inc.     

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.     

The expression of β-galactosidase by Escherichia coli during continuous culture

We have used the technique of continuous culture to study the expression of β-galactosidase in Escherichia coli. In these experiments the cultures were grown on carbon-limited media in which half of the available carbon was supplied as glycerol, glucose, or glucose 6-phosphate, and the other half as lactose. Lactose itself provided the sole source of inducer for the lac operon. The steady-state specific activity of the enzyme passed through a maximal value as a function of dilution rate. Moreover, the rate at which activity was maximal (0.40 h^?1) and the observed specific activity of the enzyme at a given growth rate were found to be identical in each of the three media tested. This result was unexpected, since the steady-state specific activity can be shown to be equal to the differential rate of enzyme synthesis, and since it is known that glycerol, glucose, and glucose-6-P-cause different degrees of catabolite repression in batch culture. The differential rate of β-galactosidase synthesis was an apparently linear function of the rate of lactose utilization per milligram protein regardless of the composition of the input medium. That is, it is independent of the rate of metabolism of substrates other than lactose which are concurrently being utilized and the enzyme level appears to be matched to the metabolic requirement for it. If this relationship is taken to indicate the existence of a fundamental control mechanism, it may represent a form of attenuation of the rate of β-galactosidase synthesis which is independent of cyclic AMP levels.     

Trehalose mobilization during early germination ofPilobolus longipes sporangiospores

Pilobolus longipes spores were activated by either exogenous glucose or 6-deoxyglucose. Trehalose content of glucose-activated spores increased and the substrate for trehalose synthesis was exogenous glucose. Addition of 6-deoxyglucose resulted in mobilization of trehalose, with about 20% of the reserve being consumed in the first hour. Little or no change in trehalase activity occurred during spore activation. Most of the trehalase activity associated with spores could be removed by washing with phosphate buffer. This extracellular enzyme was relatively stable, had a pH optimum of 5.6 and a K_m of about 0.5 mM and was estimated to be 66,000 in molecular weight. The specific activity of the crude enzyme extracts fromP. longipes was not influenced by cAMP, but, under the same conditions, the regulatory trehalase fromSaccharomyces cerevisiae became activated. These experiments indicate that trehalase activity in germinatingP. longipes spores may not be regulated by cAMP-dependent phosphorylation. Instead, the results suggest that trehalose is mobilized by a decompartmentation process.     

Experimental investigation of the external nitrification biological nutrient removal activated sludge (ENBNRAS) system

A systematic lab-scale experimental investigation is reported for the external nitrification (EN) biological nutrient removal (BNR) activated sludge (ENBNRAS) system, which is a combined fixed and suspended medium system. The ENBNRAS system was proposed to intensify the treatment capacity of BNR-activated sludge (BNRAS) systems by addressing two difficulties often encountered in practice: (a) the long sludge age for nitrification requirement; and (b) sludge bulking. In the ENBNRAS system, nitrification is transferred from the aerobic reactor in the suspended medium activated sludge system to a fixed medium nitrification system. Thus, the sludge age of the suspended medium activated sludge system can be reduced from 20 to 25 days to 8 to 10 days, resulting in a decrease in reactor volume per ML wastewater treated of about 30%. Furthermore, the aerobic mass fraction can also be reduced from 50% to 60% to <30% and concommitantly the anoxic mass fraction can be increased from 25% to 35% to >55% (if the anaerobic mass fraction is 15%), and thus complete denitrification in the anoxic reactors becomes possible. Research indicates that both the short sludge age and complete denitrification could ameliorate anoxic aerobic (AA) or low food/microorganism (F/M) ratio filamentous bulking, and hence reduce the surface area of secondary settling tanks or increase the treatment capacity of existing systems. The lab-scale experimental investigations indicate that the ENBNRAS system can obtain: (i) very good chemical oxygen demand (COD) removal, even with an aerobic mass fraction as low as 20%; (ii) high nitrogen removal, even for a wastewater with a high total kjeldahl nitrogen (TKN)/COD ratio, up to 0.14; (iii) adequate settling sludge (diluted sludge volume index [DSVI] <100 mL/g); and (iv) a significant reduction in oxygen demand.     

Role of glycogen in acetate uptake and polyhydroxyalkanoate synthesis in anaerobic-aerobic activated sludge with a minimized polyphosphate content

The role of glycogen in the uptake of acetate in anaerobic-aerobic activated sludge without enhanced biological phosphorus removal were investigated. Although the polyphosphate content of the sludge was minimized by lowering the phosphorus feeding concentration, significant acetate uptake and accumulation of polyhydroxyalkanoates (PHAs) were observed in proportion to glycogen consumption under anaerobic conditions. The results of anaerobic inhibition studies, which showed suppressive effects on acetate uptake by a glycolysis inhibitor (iodoacetate) but not by a membrane ATPase inhibitor (N,N′-dicyclohexyl carbodiimide), supported an assumption that glycogen degradation through glycolysis supplies the required ATP and reducing power for PHA synthesis from acetate and consumed glycogen. Under subsequent aerobic conditions, the accumulated PHAs were depleted and the consumed glycogen recovered to the same level as that at the start of the anaerobic phase. Iodoacetate also inhibited the recovery of glycogen under aerobic conditions, suggesting that nearly 50% of the PHAs depleted was used for glycogen synthesis through reversed glycolysis.     

Salicylic acid increased aldose reductase activity and sorbitol accumulation in tomato plants under salt stress

Increased aldose reductase (ALR) activities were detected in the leaf tissues of tomato plants grown for 3 weeks in culture medium containing 10^?7 or 10^?4 M salicylic acid (SA), and in the roots after the 10^?4 M SA pretreatment. The ALR activity changed in parallel with the sorbitol content in the leaves of the SA-treated plants. Salt stress elicited by 100 mM NaCl enhanced the accumulation of sorbitol in the leaves of control plants and as compared with the untreated control the sorbitol content in the SA-pretreated leaves remained elevated under salt stress. DEAE cellulose anionexchange column purification of the protein precipitated with 80 % (NH₄)₂SO₄ revealed two enzyme fractions with ALR activity in both the leaf and the root tissues. The fraction of the leaf extract that was not bound to the column reacted with glucose and glucose-6-P as substrates, whereas glucose was not a substrate for the bound fraction or for root isoenzymes. The root enzyme was less sensitive to salt treatment: 50 mM NaCl caused 30 % inhibition in the leaf extract, whereas the enzyme activity of the root extract was not affected. It is suggested that increased ALR activity and sorbitol synthesis in the leaves of SA-treated tomato plants may result in an improved salt stress tolerance.     

Sequential Substrate Removal in Response to Qualitative Shock Loading of Activated Sludge Systems

Heterogeneous populations acclimated to sorbitol or mannitol were subjected to shock loading with glucose during growth on the sugar alcohol. Under these severe shock-loading conditions, glucose caused immediate cessation of sorbitol or mannitol removal. Metabolism of these compounds was renewed subsequent to glucose exhaustion. Under similar shock-loading conditions, with the use of cells operationally defined as “old,” the sugar alcohols were removed concurrently with glucose. In the absence of a nitrogen source with old cells, the added glucose was removed slowly in the presence of sorbitol and not at all in the mannitol-acclimated system. These results indicate the potential variability in purification efficiency attributable to substrate interaction.     

Atypical Polyphosphate Accumulation by the Denitrifying Bacterium Paracoccus denitrificans

Polyphosphate accumulation by Paracoccus denitrificans was examined under aerobic, anoxic, and anaerobic conditions. Polyphosphate synthesis by this denitrifier took place with either oxygen or nitrate as the electron acceptor and in the presence of an external carbon source. Cells were capable of poly-β-hydroxybutyrate (PHB) synthesis, but no polyphosphate was produced when PHB-rich cells were incubated under anoxic conditions in the absence of an external carbon source. By comparison of these findings to those with polyphosphate-accumulating organisms thought to be responsible for phosphate removal in activated sludge systems, it is concluded that P. denitrificans is capable of combined phosphate and nitrate removal without the need for alternating anaerobic/aerobic or anaerobic/anoxic switches. Studies on additional denitrifying isolates from a denitrifying fluidized bed reactor suggested that polyphosphate accumulation is widespread among denitrifiers.     

Sorbitol can fuel mouse sperm motility and protein tyrosine phosphorylation via sorbitol dehydrogenase

Energy sources that can be metabolized to yield ATP are essential for normal sperm functions such as motility. Two major monosaccharides, sorbitol and fructose, are present in semen. Furthermore, sorbitol dehydrogenase (SORD) can convert sorbitol to fructose, which can then be metabolized via the glycolytic pathway in sperm to make ATP. Here we characterize Sord mRNA and SORD expression during mouse spermatogenesis and examine the ability of sorbitol to support epididymal sperm motility and tyrosine phosphorylation. Sord mRNA levels increased during the course of spermatogenic differentiation. SORD protein, however, was first detected at the condensing spermatid stage. By indirect immunofluorescence, SORD was present along the length of the flagella of caudal epididymal sperm. Furthermore, immunoelectron microscopy showed that SORD was associated with mitochondria and the plasma membranes of sperm. Sperm incubated with sorbitol maintained motility, indicating that sorbitol was utilized as an energy source. Sorbitol, as well as glucose and fructose, were not essential to induce hyperactive motility. Protein tyrosine phosphorylation increased in a similar manner when sorbitol was substituted for glucose in the incubation medium used for sperm capacitation. These results indicate that sorbitol can serve as an alternative energy source for sperm motility and protein tyrosine phosphorylation.     

Studies on the effect of inoculation of activated sludge with bacteria actively degrading hydrocarbons on the biodegradation of petroleum products

Eighteen strains of bacteria were isolated from activated sludge purifying petroleum-refining wastewaters. These strains were plated on solidified mineral medium supplemented with oil fraction in concentration 1000 mg/l. Four of the strains that grew best in the presence of oil were selected for further studies. The strains were identified based on Bonde's scheme and microscopic observations. Three of them belonged to the genus Arthrobacter and one to the genus Micrococcus. Stationary cultures of single strains and their mixtures were set up in mineral medium containing oil (sterile and non-sterile) as sole carbon source in concentration 1000 mg/l. The oils were found to be removed the most efficiently by a mixture of the strains. After 14 days of culture the amount of oil was utilized by from 63 to 95%. In the next stage of the studies the bacteria were used to inoculate activated sludge. Stationary cultures of the activated sludge were set up in mineral medium with oil. The utilisation of petroleum products by non-inoculated activated sludge (control), activated sludge inoculated with a single strain or a mixture of all four strains was examined. In both inoculated activated sludge cultures approximately 80% of the oils were removed, compared to 60% in the control activated sludge. Therefore, inoculated activated sludge showed 20% higher effectiveness of removal of petroleum derivatives.     

Bioaugmentation of Activated Sludge by an Indigenous 3-Chloroaniline-Degrading Comamonas testosteroni Strain, I2gfp

A strain identified as Comamonas testosteroni I2 was isolated from activated sludge and found to be able to mineralize 3-chloroaniline (3-CA). During the mineralization, a yellow intermediate accumulated temporarily, due to the distal meta-cleavage of chlorocatechol. This strain was tested for its ability to clean wastewater containing 3-CA upon inoculation into activated sludge. To monitor its survival, the strain was chromosomally marked with the gfp gene and designated I2gfp. After inoculation into a lab-scale semicontinuous activated-sludge (SCAS) system, the inoculated strain maintained itself in the sludge for at least 45 days and was present in the sludge flocs. After an initial adaptation period of 6 days, complete degradation of 3-CA was obtained during 2 weeks, while no degradation at all occurred in the noninoculated control reactor. Upon further operation of the SCAS system, only 50% 3-CA removal was observed. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes revealed a dynamic change in the microbial community structure of the activated sludge. The DGGE patterns of the noninoculated and the inoculated reactors evolved after 7 days to different clusters, which suggests an effect of strain inoculation on the microbial community structure. The results indicate that bioaugmentation, even with a strain originating from that ecosystem and able to effectively grow on a selective substrate, is not permanent and will probably require regular resupplementation.     

Characterization and biological treatment of pickling industry wastewater

Pickling industry wastewaters present unique difficulties in biological treatment because of high salt content (3–6% salt). Conventional activated sludge cultures disintegrate or loose microbial activity as a result of plasmolysis at salt concentrations above 1%. In order to overcome adverse effects of salt in pickling wastewater, salt tolerant bacteria (Halobacter halobium) was added to activated sludge culture and used in biological treatment of the wastewater in an activated sludge unit. After characterization and nutrient balancing of the wastewater, an activated sludge unit was used in laboratory to investigate the effects of major process variables such as sludge age and hydraulic residence time on performance of the system. Single stage and two stage activated processes were used for the treatment of the pickling wastewater. More than 95% of COD removal was obtained with a single stage process at a sludge age of θ_c=10?d and hydraulic residence time of θ_H=30?h. Similar results were obtained with the two stage process, when sludge ages and hydraulic residence times for each stage were θ_c1=θ_c2=10?d, and θ_H1=θ_H2=15?h, respectively. Kinetic coefficients were determined and the design equations were developed by using the experimental data.     

The ’glucose effect’ in callus cultures ofNicotiana plmmbaginifolia is enzyme specific

We have studied the effect of glucose on different enzyme activities in callus cultures of N.plumbaginifolia. We provided evidence that the increase in glucose concentration represses the synthesis of GDH, AcPh and MDH whereas ADH and LDH are unaffected. When glucose repressed cultures were transferred to the low glucose concentration medium the activity of GDH and AcPh resumed to the level shown by non repressed cultures. Sucrose and fructose were as effective as glucose in repressing GDH activity. These results support the hypothesis of the existence of a catabolite dependent regulation of enzyme synthesis in higher plants.     

Thermostable amylolytic enzymes from a cellulolytic fungusMyceliophthora thermophila D14 (ATCC 48 104)

Summary The production of amylolytic enzymes by a thermophilic cellulolytic fungus,Myceliophthora thermophila D14 was investigated by batch cultivation in Czapek-Dox medium at 45° C. Among various nitrogenous compounds used, NaNO₃ and KNO₃ were found to be the best for amylase production. Starch, cellobiose and maltose induced the synthesis of amylase while glucose, fructose, galactose, lactose, arabinose, xylose, sorbitol, mesoinositol and sucrose did not. Calcium ions had the most stimulating effect on enzyme formation amongst many ions investigated. The synthesis of amylolytic enzymes was dependent on growth and occurred predominantly in the mid-stationary phase. The enzyme was active in a broad temperature range (50° C–60° C) and displayed activity optima at 60° C and pH 5.6.     

Enzymatic activity in the activated-sludge floc matrix

The enzymatic activity of activated sludge was investigated with special emphasis on the localization of the enzymes in the sludge floc matrix. Activated sludge from an advanced activated-sludge treatment plant, performing biological N and P removal, was used. An enzymatic fingerprint was established using a panel of six different enzymes. The fingerprint revealed peptidase as the most dominating specific enzyme tested. By monitoring sludge bulk enzymatic activity over a 3-month period using fluorescein diacetate as an enzyme substrate, considerable variations in activity were observed even over short periods (a few days). The variation in esterase activity was to some extent correlated to the presence of humic compounds in the sludge, but not to the sludge protein content. Comparison of full sludge enzyme activity to the activity of a batch-grown sludge culture indicated that enzymes accumulated in sludge flocs. A large proportion of the exoenzymes were immobilized in the sludge by adsorption in the extracellular polymeric substances (EPS) matrix. This was demonstrated by extraction of EPS from the activated sludge using cation exchange. Contemporary to the release of EPS a very large fraction of the exoenzymes was released into the water. This showed that the exoenzymes should be considered to be an integrated part of the EPS matrix rather than as direct indicators of the microbial activity or biomass.     

Conversion of glucose to sorbitol and fructose by liver-derived cells in culture.

Conversion of glucose to fructose and sorbitol is documented in rat hepatoma-derived cultured cells (HTC cells). After addition of 5.5 mM [U-14C]glucose to incubation medium, labeled sorbitol and fructose accumulated intracellularly at a linear rate over a period of 60 min. The sugars were isolated, identified, and quantitated by paper chromatography, gas-liquid chromatography, and enzymatic phosphorylation of fructose. Primary culture of adult rat hepatocytes was analyzed similarly and demonstrated no significant accumulation of labeled fructose or sorbitol. The basis for this difference between HTC cells and primary hepatocyte culture was examined both in terms of enzyme activities that mediate the formation of sorbitol and fructose and in terms of the catabolism of these sugars. Both types of culture (as well as extracts of intact rat liver) exhibited enzymatic activities catalyzing the conversion of glucose to sorbitol (aldose reductase) and sorbitol to fructose (sorbitol dehydrogenase). However, the cultures differed strikingly with regard to the catabolism of sorbitol and fructose. The conversion of labeled sorbitol to metabolites in HTC cells was negligible; by contrast, hepatocytes in primary culture utilized the sugars at rates comparable to that of glucose, which may account for the lack of their accumulation in primary culture. The findings suggest that the conversion of glucose to sorbitol and fructose by HTC cells may represent a retained normal liver function, one which is amplified by the inability of HTC cells to dispose of these sugars.     

Induction and carbon catabolite repression of isoamylase production in Rhizopus oryzae PR7

The growth and the extracellular isoamylase production by Rhizopus oryzae PR7 MTCC 9642 were studied in a stationary culture at 28°C, with maximum isoamylase production obtained after 72 hours. Glycogen was found to be the best inducer for isoamylase synthesis, followed by maltose and dextrin. The enzyme was found to be repressed by glucose and this repression was not overcome by the addition of cGMP. The abrupt reduction in enzyme synthesis after the addition of exogenous glucose in a glycogen-induced culture medium confirmed the repressive action of glucose. An almost similar rate of repression was found to be exerted by α- and β-cyclodextrins. The inhibition of enzyme production after the addition of cycloheximide, a translation blocker, indicated the existence of de novo synthesis of the enzyme.