Use of microbial activity parameters for determination of a biosolid stability index

Variations in microbial activity during the aerobic digestion of sludge generated at wastewater treatment plants were studied. Results obtained by the measurement of enzymatic activity and microbiological parameters were compared with those determined by traditional methods (COD, suspended solids, etc.). Their variation with digestion time was monitored for batch digestion over a period of 135 days. The relationship between these measurements and control parameters of the sludge was also investigated. It was found that the traditional physicochemical and microbiological parameters present a series of problems which detract from their usefulness. The enzymatic parameters dehydrogenase activity (primary metabolism) and esterase activity (secondary metabolism) are better able to characterise the process, and the ratio of these two variables may be used to estimate the degree of endogenesis and, consequently, the degree of stability of the aerobic sludge digestion. In addition, these techniques are swift and simple to employ.     

Correlation between the activity of enzymes involved in energy and carbohydrate metabolism with the size and weight parameters of some coregonidae and salmonidae fish

The correlation between the energy and carbohydrate metabolism rate with the length and weight of salmon (Salmo salar L.) and whitefish (Coregonus lavaretus L., Coregonus albula L.) studied in nature and farmed rainbow trout (Parasalmo mykiss Walb.) has been investigated. The results of the study show that the largest and fastest growing fish had high activity of enzymes involved in aerobic energy metabolism (cytochrome c oxidase and malate dehydrogenase) and anaerobic metabolism (lactate dehydrogenase in muscles and 1-glycerophosphate dehydrogenase and glucose-6-phosphate dehydrogenase in liver). Age-related changes of the investigated parameters are caused by a general reduction of the aerobic metabolism rate and oxygen consumption during ontogenesis, increased lipid metabolism, and the amount of stored substances. It is also shown that pubescent male and female whitefish (4+ and 5+) have differences in the correlation between enzyme activity of cytochrome c oxidase, 1-glycerophosphate and glucose-6-phosphate dehydrogenases and the length and weight of individuals.     

Microbiological parameters as an additional tool to improve wastewater treatment plant monitoring

The operation of an activated sludge wastewater treatment plant (WWTP) was assessed by determining the removal efficiency of treatment processes. For that purpose, different physicochemical and microbiological parameters, including total, active and culturable bacteria, were measured along a year. Values of both physicochemical and microbiological parameters were significantly reduced by biological treatment but no relation between these groups of parameters was obtained. Besides, results suggest that microbiological parameters were more sensitive than physicochemical ones to changes in WWTP conditions (i.e. influent quality). Additionally, high percentages of noncultured (>88%) and noncultured remaining active (until 50%) cells were detected before and after treatment. Therefore, ISO standards adopted to enumerate indicator bacteria by Spanish legislation, and based on culturability, underestimate the bacterial population present in wastewater. We have compared two culture-independent methods for bacterial enumeration, microscopy and flow cytometry (FCM) and results indicate that FCM provides a rapid and automated quantification in WWTP. We conclude that microbiological parameters are essential to monitor the correct WWTP operation and we propose quantification of active bacteria as indicator of wastewater microbiological quality.     

Study of biochemical and microbiological parameters during composting of pine and eucalyptus bark

To study the possibility of some residues from pulp and paper industry being used as substrates to produce seedlings in containers, three composting experiments were carried out using eucalyptus bark, pine bark and a mixture (60:40, v:v) of pine bark+eucalyptus bark. Biochemical parameters studied were: acid and alkaline phosphatases, lipase (C10), protease, urease, beta-glucosidase and total cellulases. The microbiological populations of total aerobic bacteria, total fungi, actinomycetes, nitrifying bacteria, cellulolytic bacteria and fungi were also evaluated. At the end of the process physicochemical characterization of composts was also performed. Results showed in general that the highest microbiological populations as well as for enzymatic activities occurred during the thermophilic phase (>40 degrees C) of the process. On the other hand and according to the physicochemical characteristics of composts pine bark is the most appropriate material to be used in the formulation of substrates to produce plants in containers.     

Heavy metal fractionation and organic matter stabilization in sewage sludge treatment wetlands

The presence of heavy metals in sludge stabilized in a reed bed system may affect its use for agricultural purposes. However, the environmental impact of sludge depends on the availability and phytotoxicity of these heavy metals.The aim of this research was to determine the effectiveness of a reed bed (Phragmites australis) sludge treatment system in two urban wastewater treatment plants in Italy after a three-year period of operation: (i) by estimating the process of sludge stabilization, following conventional and nonconventional parameters related to the evolution of organic matter quality (water soluble carbon, dehydrogenase activity, pyrolytic fragments); (ii) by following the heavy metal bioavailability in the sludge through their fractionation. For heavy metal fractionation, the Community Bureau of Reference (BCR) was followed.The results showed that there was mineralization and stabilization of sludge over time, suggested by the decrease of about 35% in water soluble carbon and of about 60-80% of dehydrogenase activity. Moreover, significant values of benzene (17%), toluene (31%) and phenol (9%) were found at the end of experimentation in both treatment wetlands, highlighting the re-synthesis of humic-like matter.The results also showed that the content of heavy metals after 30 months was associated with the less mobile fractions of the sludge (more than 60% of total heavy metal content for almost metal), in particular, the fraction linked to the organic matter.     

Bacterial survival and association with sludge flocs during aerobic and anaerobic digestion of wastewater sludge under laboratory conditions.

The fate of indicator bacteria, a bacterial pathogen, and total aerobic bacteria during aerobic and anaerobic digestion of wastewater sludge under laboratory conditions was determined. Correlation coefficients were calculated between physical and chemical parameters (temperature, dissolved oxygen, pH, total solids, and volatile solids) and either the daily change in bacterial numbers or the percentage of bacteria in the supernatant. The major factor influencing survival of Salmonella typhimurium and indicator bacteria during aerobic digestion was the temperature of sludge digestion. At 28 degrees C with greater than 4 mg of dissolved oxygen per liter, the daily change in numbers of these bacteria was approximately -1.0 log10/ml. At 6 degrees C, the daily change was less than -0.3 log10/ml. Most of the bacteria were associated with the sludge flocs during aerobic digestion of sludge at 28 degrees C with greater than 2.4 mg of dissolved oxygen per liter. Lowering the temperature or the amount of dissolved oxygen decreased the fraction of bacteria associated with the flocs and increased the fraction found in the supernatant.     

Effect of aeration intensity on the biochemical composition of baker's yeast. II. Activities of the oxidative enzymes

When the effect of catabolite repression is eliminated Saccharomyces cerevisiae prefers an aerobic metabolism. The potential for completely aerobic catabolism exists even in circumstances where its action is limited by the oxygen available. When the oxygen absorption in the medium is adequate, yeast uses a solely oxidative metabolism for energy-yielding reactions. The changes observed in the activity of malate dehydrogenase can be described as a function of two isoenzymes, both of which are affected by oxygen; the isoenzyme participating in the glyoxylate cycle shows variations in activity similar to that observed in isocitrate lyase. NAD-linked glutamate dehydrogenase activity roughly follows that of malate dehydrogenase and isocitrate lyase; in cultivations with the same growth rate the NADP-linked dehydrogenase is insensitive to the oxygen level. The cytochromes aa₃, b, and c have a clear maximum at low oxygen tension, the most sensitive being cytochrome aa₃. The imbalance between cytochrome c:oxygen oxidoreductase activity and the amount of cytochrome aa₃, and the correlation observed between respiration rate and the activities of cytochrome c oxidase and NADH₂:cytochroine c oxidoreductase are discussed. Methods used for estimation of cytochromes are compared.     

Comparison of microbial and meiofaunal community analyses for determining impact of heavy metal contamination

The impact of long-term heavy metal contamination on soil communities was assessed by a number of methods. These included plate counts of culturable bacteria, community level physiological profiling (CLPP) by analysis of the utilization of multiple carbon sources in BIOLOG plates, community fatty acid methyl ester (C-FAME) profiling and dehydrogenase enzyme activity measurements. These approaches were complemented with microscopic assessments of the diversity of the nematode community. Samples from two sites with different histories of heavy-metal input were assessed. Major differences in microbial and meiofaunal parameters were observed both between and within the sites. There was a large degree of congruence between each of the microbiological approaches. In particular, one sample appeared to be distinguished by a reduction in culturable bacteria (especially pseudomonads), limited response to carbon sources in CLPP, and major differences in extracted fatty acid profiles. The use of multivariate analysis to examine the relationship between microbial and physicochemical measurements revealed that CLPP and plate counts were useful for determining the gross effect of metals on soil microbial communities, whereas proportions of metal-resistant bacteria and dehydrogenase activity differentiated between the two sites. Copper and zinc concentrations and pH all showed significant correlation with the microbial parameters. Nematode community structure was affected to a greater extent by soil pH than by metal content, but the within-site rankings were the same as those achieved for microbiological analyses. The use of these methods for field evaluation of the impact of industrial pollution may be possible provided care is taken when interpreting the data.     

Changes induced by ischemia on some cerebral enzymatic activities related to energy transduction and amino acid metabolism

The maximal rate of some cerebral enzymatic activities related to energy transduction (hexokinase; phosphofructokinase; lactate dehydrogenase; citrate synthase; malate dehydrogenase; total NADH-cytochrome c reductase; cytochrome oxidase), amino acid metabolism (glutamate decarboxylase; glutamate dehydrogenase) and cholinergic metabolism (acetylcholine esterase) were tested in the cerbral cortex and in sub-cortical area of rats. The evaluations were performed both in the homogenate in toto and in the crude mitochondrial fraction, before and after a postdecapitative normothermic ischemia of 5, 10, 20, and 40 min duration. The results are discussed also with respect to the pharmacological pretreatment with two biological substances which may modulate amino acid (l-alanine) and phospholipid metabolism (CDP-choline). The analysis of the present data suggests the occurrence in brain tissue of a variety of interrelated factors implicated in the ischemia-induced changes of the maximal rate of the enzymatic activities related to the energy transduction. These include: (a) rearrangement of the enzymatic activities because of the changed metabolic and chemico-physical condition; (b) decrease in the activity of enzymes related to the electron transfer chain and glycolysis; (c) changes in enzymes related to mitochondrial membranes. The effects of in vivo administration of alanine or CDP-choline, even if significant, are not consistent throughout the time period studied.     

Aerobic and two-stage anaerobic-aerobic sludge digestion with pure oxygen and air aeration

The degradability of excess activated sludge from a wastewater treatment plant was studied. The objective was establishing the degree of degradation using either air or pure oxygen at different temperatures. Sludge treated with pure oxygen was degraded at temperatures from 22 degrees C to 50 degrees C while samples treated with air were degraded between 32 degrees C and 65 degrees C. Using air, sludge is efficiently degraded at 37 degrees C and at 50-55 degrees C. With oxygen, sludge was most effectively degraded at 38 degrees C or at 25-30 degrees C. Two-stage anaerobic-aerobic processes were studied. The first anaerobic stage was always operated for 5 days HRT, and the second stage involved aeration with pure oxygen and an HRT between 5 and 10 days. Under these conditions, there is 53.5% VSS removal and 55.4% COD degradation at 15 days HRT - 5 days anaerobic, 10 days aerobic. Sludge digested with pure oxygen at 25 degrees C in a batch reactor converted 48% of sludge total Kjeldahl nitrogen to nitrate. Addition of an aerobic stage with pure oxygen aeration to the anaerobic digestion enhances ammonium nitrogen removal. In a two-stage anaerobic-aerobic sludge digestion process within 8 days HRT of the aerobic stage, the removal of ammonium nitrogen was 85%.     

Energy metabolism in the tropical abalone, Haliotis asinina Linné: Comparisons with temperate abalone species

The abalone, Haliotis asinina, is a large, highly active tropical abalone that feeds at night on shallow coral reefs where oxygen levels of the water may be low and the animals can be exposed to air. It is capable of more prolonged and rapid exercise than has been reported for temperate abalone. These unusual behaviours raised the question of whether H. asinina possesses enhanced capacities for aerobic or anaerobic metabolism. The blood oxygen transport system of H. asinina resembles that of temperate abalone in terms of a large hemolymph volume, similar hemocyanin concentrations, and in most hemocyanin oxygen binding properties; however, absence of a Root effect appears confined to hemocyanin from H. asinina and may assist oxygen uptake when hemolymph pH falls during exercise or environmental hypoxia. During exposure to air, H. asinina reduces oxygen uptake by at least 20-fold relative to animals at rest in aerated seawater, and there is no significant ATP production from anaerobic glycolysis or phosphagen hydrolysis in the foot or adductor muscles. This slowing of metabolism may contribute to survival at lower water oxygen levels than normally encountered by most temperate abalone. While crawling speeds of H. asinina in water are not exceptionally high, an aerobic expansibility of 5.5-fold at speeds less than 20% of maximum is more than 2.7-fold greater than reported for several temperate abalone. The high aerobic expansibility also supports the enhanced frequency and duration of flipping behaviour without recourse to the additional inputs from anaerobic glycolysis required by other abalone. Metabolic profiles of foot and adductor muscles of H. asinina are similar to those of other abalone. Common features are low activities of enzymes unique to aerobic ATP production, relatively high activities of arginine kinase, tauropine and d-lactate dehydrogenase as the predominant pyruvate reductases, and low intracellular pH buffering capacities. It is concluded that the exceptional abilities of H. asinina for prolonged and rapid exercise are supported by higher rates of aerobic metabolism rather than any enhanced capacity for anaerobic muscle work. It is unexpected, and instructive, that the exceptional aerobic expansibility is not apparent in obvious adjustments of the blood oxygen delivery system or muscle properties associated with aerobic ATP production. The absence of a hemocyanin Root effect, and the extent to which both aerobic and anaerobic metabolism can be reduced may be special features that assist prolonged exercise and survival of H. asinina when environmental oxygen becomes limiting.     

Lymphocyte and neutrophil cytochemistry in the dynamics of different forms of diphtheria in adults]

The cytochemical study of lymphocytes and neutrophils isolated from fractionated blood of diphtheria patients and carriers has revealed that a decrease in the activity of lymphocyte succinic dehydrogenase and myeloperoxidase can be observed at all periods of the disease and in all its forms. A decrease in the activity of lymphocyte nonspecific esterase has been noted only in patients with toxic and subtoxic diphtheria and a decrease in the activity of neutrophil alkaline phosphatase, in carriers. The analysis of correlations between the parameters of five enzymes under study (lymphocyte succinic dehydrogenase, lymphocyte acid phosphatase, lymphocyte non-specific esterase, myeloperoxidase, neutrophil alkaline phosphatase) and enzymatic rosette parameters has been made. The analysis has revealed an essential increase in the number of correlations in comparison with donors, changes in the qualitative nature of these correlations and sometimes the reversion of the correlations. Carriers have shown the greatest number of correlations. By the end of the terms of observations no restoration of normal correlations has been observed.     

Localization and catalytic properties of lactate dehydrogenase in different sperm models

A study of the intracellular localization and catalytic properties (effects of substrates and products) on lactate dehydrogenase has been carried out on a series of spermatozoa endowed with mitochondria and characterized by aerobic metabolism of fatty acids (sea urchin); aerobic metabolism of fatty acids and exogenous carbohydrates and lactic acid (bull); metabolism of glycogen connected with mitochondria (Lebistes); metabolism of periaxonemal glycogen (Octopus). The data obtained indicate that LDH is present only when carbohydrates are metabolized; its localization follows the glycogen stores; in the Octopus sperm where mitochondria are poorly developed relative to the flagellar length, and exogenous lactate is not available, LDH catalyses predominantly the reduction of pyruvate; in Lebistes and trout sperms, where mitochondria are better developed, LDH is more similar to the mammalian heart and sperm isozymes.     

Effect of Modified Atmosphere Composition on the Metabolism of Glucose by Brochothrix thermosphacta

The influence of atmosphere composition on the metabolism of Brochothrix thermosphacta was studied by analyzing the consumption of glucose and the production of ethanol, acetic and lactic acids, acetaldehyde, and diacetyl-acetoin under atmospheres containing different combinations of carbon dioxide and oxygen. When glucose was metabolized under oxygen-free atmospheres, lactic acid was one of the main end products, while under atmospheres rich in oxygen mainly acetoin-diacetyl was produced. The proportions of the total consumed glucose used for the production of acetoin (aerobic metabolism) and lactic acid (anaerobic metabolism) were used to decide whether aerobic or anaerobic metabolism predominated at a given atmosphere composition. The boundary conditions between dominantly anaerobic and aerobic metabolisms were determined by logistic regression. The metabolism of glucose by B. thermosphacta was influenced not only by the oxygen content of the atmosphere but also by the carbon dioxide content. At high CO₂ percentages, glucose metabolism remained anaerobic under greater oxygen contents.     

Light-limitation on predator-prey interactions: consequences for metabolism and locomotion of deep-sea cephalopods

The present study attempts to correlate the metabolism and locomotory behavior of 25 species of midwater Cephalopoda from California and Hawaii with the maximal activities of key metabolic enzymes in various locomotory muscle tissues. Citrate synthase (CS) and octopine dehydrogenase (ODH) activities were used as indicators of aerobic and anaerobic metabolic potential respectively. CS activity in mantle muscle is highly correlated with whole-animal rates of oxygen consumption, whereas ODH activity in mantle muscle is significantly correlated with a species' ability to buffer the acidic end-products of anaerobic metabolism. Both CS and ODH activities in mantle muscle declined strongly with a species' habitat depth. For example, CS and ODH activities ranged respectively from 0.04 units g(-1) and 0.03 units g(-1) in the deep-living squid Joubiniteuthis portieri, to 8.13 units g(-1) and 420 units g(-1) in the epipelagic squid Sthenoteuthis oualaniensis. The relationships between enzymatic activities and depth are consistent with similar patterns observed for whole-animal oxygen consumption. This pattern is believed to result from a relaxation, among deep-living species, in the need for strong locomotory abilities for visual predator/prey interactions; the relaxation is due to light-limitation in the deep sea. Intraspecific scaling patterns for ODH activities may, for species that migrate ontogenetically to great depths, reflect the counteracting effects of body size and light on predator-prey detection distances. When scaled allometrically, enzymatic activities for the giant squid, Architeuthis sp., suggest a fairly active aerobic metabolism but little burst swimming capacity. Interspecific differences in the relative distributions of enzymatic activities in fin, mantle, and arm tissue suggest an increased reliance on fin and arm muscle for locomotion among deep-living species. We suggest that, where high-speed locomotion is not required, more efficient means of locomotion, such as fin swimming or medusoid arm propulsion, are more prevalent.     

Oxygen- and Glucose-Dependent Regulation of Central Carbon Metabolism in Pichia anomala

We investigated the regulation of the central aerobic and hypoxic metabolism of the biocontrol and non-Saccharomyces wine yeast Pichia anomala. In aerobic batch culture, P. anomala grows in the respiratory mode with a high biomass yield (0.59 g [dry weight] of cells g of glucose⁻¹) and marginal ethanol, glycerol, acetate, and ethyl acetate production. Oxygen limitation, but not glucose pulse, induced fermentation with substantial ethanol production and 10-fold-increased ethyl acetate production. Despite low or absent ethanol formation, the activities of pyruvate decarboxylase and alcohol dehydrogenase were high during aerobic growth on glucose or succinate. No activation of these enzyme activities was observed after a glucose pulse. However, after the shift to oxygen limitation, both enzymes were activated threefold. Metabolic flux analysis revealed that the tricarboxylic acid pathway operates as a cycle during aerobic batch culture and as a two-branched pathway under oxygen limitation. Glucose catabolism through the pentose phosphate pathway was lower during oxygen limitation than under aerobic growth. Overall, our results demonstrate that P. anomala exhibits a Pasteur effect and not a Crabtree effect, i.e., oxygen availability, but not glucose concentration, is the main stimulus for the regulation of the central carbon metabolism.     

Strain variation in Hymenolepis diminuta: enzyme profiles

In comparative study of respiratory metabolism, it was established that the relative proportions of respiratory end-products (succinic, acetic and lactic acids) differed consistently in two strains of Hymenolepis diminuta (Toronto and ANU). The ANU strain produced more lactic acid and less succinic acid under aerobic and anaerobic conditions. In the shift from aerobic to anaerobic conditions both strains compensated by increasing their outputs of succinic acid. The ANU strain possessed significantly higher activities of hexokinase, pyruvate kinase, lactate dehydrogenase, cytosolic and mitochondrial malic enzyme and cytosolic α-glycerophosphate dehy drogenase. The Toronto strain had significantly higher activities of fumarase, succinate dehydrogenase, and fumarate reductase. There were no significant differences in the activities of phosphoenolpyruvate carboxykinase and malic dehydrogenase between strains. The fumarase activity in the Toronto strain was 16 times that of the ANU strain, its K_m (malate) was 0.8mM, as opposed to 2.5 mM, and it was less sensitive to inhibition by NAD or ATP. These observations are consistent with the patterns of end-product formation in the two strains. Ratios of end-products and calculations of approximate redox balance suggest that the Toronto strain may have a greater capacity for aerobic metabolism.     

Distinctive Features of the Functioning of Lactic Acid and the Alcohol Fermentation Enzyme in Sorghum and Pea Leaves under the Conditions of Oxygen Deficiency

Dynamic changes in the enzymatic activity of lactate dehydrogenase, L-lactate-cytochrome-c-coxidoreductase, and alcohol dehydrogenase were analyzed in the seedlings of sorghum, a plant resistant to oxygen deficiency, and peas, a plant vulnerable to oxygen deficiency, cultivated under the conditions of flooding. The subcellular localization and isoform composition of the enzymes were characterized. The mechanism underlying the adaptive reaction of cell metabolism was devised from analysis of the results obtained. The reaction is supposed to involve lactate dehydrogenase and L-lactate-cytochrome-c-oxidoreductase enzymes that suppress cytoplasm acidification in sorghum and pea roots in the case of flooding.