Total Anoxia Research Articles

The study of plant adaptation to oxygen deficiency in Saint Petersburg University

The first studies on plant anaerobiosis started at the Department of Plant Physiology at St. Petersburg University in the beginning of the XXth century, but interest in this subject became most intensive during the investigations of the ecological plant physiology group under the supervision of Prof. T. V. Chirkova. Their first step was focused on the mechanisms of transport of gases from the aerated aboveground part of the plant to the flooded root system. Further interest shifted towards clarifying the biochemistry of respiratory metabolism, pathways of reoxidation of the reduced cofactors, and protein and lipid metabolism of plants under anoxic conditions. The group’s studies have always distinguished the comparative approach, in which the changes taking place in plants differing in resistance to oxygen deficiency were analyzed. In many ways, this research was pioneering and was recognized throughout the world. For the first time the possibility of hydrogen peroxide formation in plants under total anoxia was demonstrated. The role of cell membranes in adaptation processes was revealed. Pioneering investigations distinguished the features of photosynthesis in an oxygen-free environment and the work of an antioxidant system under conditions of anoxia and post-anoxic oxidative effects. Now, the plant ecophysiology group of the Department of Plant Physiology and Biochemistry of St. Petersburg State University concentrates on the mechanisms of anaerobic signal transduction and reveals how plant hormones regulate adaptation to anoxic and post-anoxic stresses.

Remodelling of microRNAs in colorectal cancer by hypoxia alters metabolism profiles and 5-fluorouracil resistance.

Solid tumours have oxygen gradients and areas of near and almost total anoxia. Hypoxia reduces sensitivity to 5-fluorouracil (5-FU)-chemotherapy for colorectal cancer (CRC). MicroRNAs (miRNAs) are hypoxia sensors and were altered consistently in six CRC cell lines (colon cancer: DLD-1, HCT116 and HT29; rectal cancer: HT55, SW837 and VACO4S) maintained in hypoxia (1 and 0.2% oxygen) compared with normoxia (20.9%). CRC cell lines also showed altered amino acid metabolism in hypoxia and hypoxia-responsive miRNAs were predicted to target genes in four metabolism pathways: beta-alanine; valine, leucine, iso-leucine; aminoacyl-tRNA; and alanine, aspartate, glutamate. MiR-210 was increased in hypoxic areas of CRC tissues and hypoxia-responsive miR-21 and miR-30d, but not miR-210, were significantly increased in 5-FU resistant CRCs. Treatment with miR-21 and miR-30d antagonists sensitized hypoxic CRC cells to 5-FU. Our data highlight the complexity and tumour heterogeneity caused by hypoxia. MiR-210 as a hypoxic biomarker, and the targeting of miR-21 and miR-30d and/or the amino acid metabolism pathways may offer translational opportunities.

Hypoxia-inducible factor-1 mediates adaptive developmental plasticity of hypoxia tolerance in zebrafish, Danio rerio.

In recent years, natural and anthropogenic factors have increased aquatic hypoxia the world over. In most organisms, the cellular response to hypoxia is mediated by the master regulator hypoxia-inducible factor-1 (HIF-1). HIF-1 also plays a critical role in the normal development of the cardiovascular system of vertebrates. We tested the hypothesis that hypoxia exposures which resulted in HIF-1 induction during embryogenesis would be associated with enhanced hypoxia tolerance in subsequent developmental stages. We exposed zebrafish (Danio rerio) embryos to just 4 h of severe hypoxia or total anoxia at 18, 24 and 36 h post-fertilization (hpf). Of these, exposure to hypoxia at 24 and 36 hpf as well as anoxia at 36 hpf activated the HIF-1 cellular pathway. Zebrafish embryos that acutely upregulated the HIF-1 pathway had an increased hypoxia tolerance as larvae. The critical window for hypoxia sensitivity and HIF-1 signalling was 24 hpf. Adult male fish had a lower critical oxygen tension (Pcrit) compared with females. Early induction of HIF-1 correlated directly with an increased proportion of males in the population. We conclude that mounting a HIF-1 response during embryogenesis is associated with long-term impacts on the phenotype of later stages which could influence both individual hypoxia tolerance and population dynamics.

Integrated ichnology and ichthyology of the Oligocene Menilite Formation, Skole and Subsilesian nappes, Polish Carpathians: A proxy to oxygenation history

The anoxic, mostly black or brown fine-grained sediments of the Menilite Formation (Oligocene–Early Miocene) in the Skole and Subsilesian nappes contain thin layers of bioturbated green or grey-green mudstones, some of which contain the trace fossils Halimedides annulata, Multina isp., Palaeophycus ?tubularis, ?Planolites isp., Rhizocorallium isp., Trichichnus isp. and Zoophycos isp. The Trichichnus–Palaeophycus–(Multina, Halimedides)–Rhizocorallium suite indicates an increase in oxygenation of sediments. The contribution of different ecological groups of fishes, including epipelagic, bathypelagic, benthopelagic, neritic and reef, and demersal taxa changes significantly through the Menilite Formation. The absence or reduction of bathypelagic fishes points to anoxia in the water column. The combination of ichnological and ichthyological data and incorporation of data on benthic foraminifers allowed a reconstruction of oxygenation changes in the sediment and water column during deposition of the Menilite Formation. Total anoxia at the sea floor and in the water column, attributed to a combination of thermo-stratification and extremely high organic productivity, occurred only during the period reflected by ichthyofaunal Zone IPM2 (middle part of the NP23 Zone). Anoxia restricted to the basin floor or upper slope, related in part to upwelling, occurred during sedimentation of the upper part of the Menilite Formation.

Heat Perfusion for Malignancy

Hyperthermia, total anoxia and low pH have shown selective lethal effect on malignant cells. A perfusion system was devised to combine these modalities and was tried in 4 cases of advanced malignancy. A perfusion lasting for 45 minutes was found safe for normal tissue and yet selectively injurious to malignant cells. Technically this is a very simple procedure and its principles can be utilized in regional infusion also. Though the clinical material is scanty,the method deserves further trial.

DETERMINATION OF RESPIRATORY PARAMETERS OF ONION BULBS (ALLIUM CEPA L.) UNDER TOTAL ANOXIA

This work aimed to investigate the effect of total anoxia (100 N 2 ) on the respiratory parameters of onion bulbs. Freshly harvested onion bulbs (Allium cepa L. 'Rouge Amposta' obtained from organic farming) were used for this experiment. Under total anoxia, Fermentative Index (FI) values were 0.048, 0.072 and 0.14 mmol CO 2 kg -1 h -1 at 4, 10 and 20°C, respectively. The fermentative process was temperature dependent and followed an Arrhenius-like equation (R 2 =0.92). The Q 10 of onion bulbs under anoxia was 1.93 and FI 0 (fermentative index at 0°C) was estimated at 0.037 mmol CO 2 kg -1 h -1 with an activation energy (Ec) of 44 578 J mol -1 . Moreover, the FI of the bulbs (in mmol CO 2 kg -1 h -1 ) was measured within 6h after closing the vessels (jars). Beyond 24h at 20°C, high CO 2 production was observed and was likely due to anaerobic or aero/anaerobic microorganisms such as yeast and lactic acid bacteria. Furthermore, ethanol production under anoxia was also estimated and averaged <0.001, 0.55 and 0.79 pmol kg -1 h -1 at 4, 10 and 20°C, respectively. These results indicated that onion bulbs are less sensitive to anoxia and tolerated total anoxia for at least 24 h.

Scandinavian Clinical practice guidelines for therapeutic hypothermia and post‐resuscitation care after cardiac arrest

Sudden cardiac arrest survivors suffer from ischaemic brain injury that may lead to poor neurological outcome and death. The reperfusion injury that occurs is associated with damaging biochemical reactions, which are suppressed by mild therapeutic hypothermia (MTH). In several studies MTH has been proven to be safe, with few complications and improved survival, and is recommended by the International Liaison of Committee on Resuscitation. The aim of this paper is to recommend clinical practice guidelines for MTH treatment after cardiac arrest from the Scandinavian Society of Anaesthesiology and Intensive Care Medicine (SSAI). Relevant studies were identified after two consensus meetings of the SSAI Task Force on Therapeutic Hypothermia (SSAITFTH) and via literature search of the Cochrane Central Register of Controlled Trials and Medline. Evidence was assessed and consensus opinion was used when high-grade evidence (Grade of Recommendation, GOR) was unavailable. A management strategy was developed as a consensus from the evidence and the protocols in the participating countries. Although proven beneficial only for patients with initial ventricular fibrillation (GOR A), the SSAITFTH also recommend MTH after restored spontaneous circulation, if active treatment is chosen, in patients with initial pulseless electrical activity and asystole (GOR D). Normal ethical considerations, premorbid status, total anoxia time and general condition should decide whether active treatment is required or not. MTH should be part of a standardized treatment protocol, and initiated as early as possible after indication and treatment have been decided (GOR E). There is insufficient evidence to make definitive recommendations among techniques to induce MTH, and we do not know the optimal target temperature, duration of cooling and rewarming time. New studies are needed to address the question as to how MTH affects, for example, prognostic factors.

Uncoupling Proteins 2 and 3 Function in Concert to Augment Tolerance to Cardiac Ischemia

Transient cardiac ischemia activates cell survival signaling, conferring subsequent ischemia tolerance to the heart. This biological phenomenon, termed ischemic preconditioning, results in improved clinical outcome and attenuated infarct size following myocardial infarction. To explore genomic modifications underpinning this ischemia tolerance, we delineated the regulation and function of the cardiac enriched mitochondrial uncoupling proteins 2 and 3 during delayed ischemic preconditioning in the rat. Cardiac transcripts of genes encoding uncoupling proteins 2 and 3 are up-regulated in parallel with infarct size reduction in preconditioned hearts. Mitochondria isolated from preconditioned hearts exhibit an augmented inducible proton leak. In parallel, following anoxia-reoxygenation these mitochondria generate less hydrogen peroxide compared with non-preconditioned mitochondria. Preconditioning in rat cardiac derived myoblasts is abolished following uncoupling protein-2 depletion by RNA-interference. RNAi of uncoupling protein-3 partially attenuates the capacity to precondition these cells. Functional characterization of anoxia and reoxygenation tolerance following uncoupling protein 2 or 3 and combined 2 and 3 RNAi shows the largest reduction in viability follows depletion of both homologues. Uncoupling protein-2 depletion alone significantly attenuates anoxia-reoxygenation tolerance but uncoupling protein-3 depletion does not reduce anoxia tolerance. In parallel combined uncoupling protein depletion and isolated uncoupling protein-2 depletion augments ROS production in viable cardiomyocytes following anoxia-reoxygenation. Concurrent anti-oxidant administration ameliorates the uncoupling protein-depleted anoxia-susceptible phenotype. In conclusion, mitochondrial uncoupling proteins are necessary components of ischemia tolerance and function as components of the cellular antioxidant defense program. In the cytoprotective hierarchy, uncoupling protein-2 appears to play a greater role than uncoupling protein-3 in modulating ischemia/anoxia tolerance in heart-derived cells.

The extinction by sulfide—turnover and recovery of a naturally eutrophic, meromictic seawater lake

Since 1994 seasonal variations of temperature, salinity and vertical distribution of dissolved oxygen, nutrients, dissolved organic carbon (DOC), surface-active substances (SAS), reduced sulfur species (RSS), phyto- and zooplankton have been investigated in water column of the Rogoznica Lake. During the thermohaline stratification (spring and summer), the surface water is well-oxygenated while anoxia is occurring in the bottom layer. Anoxic deep water is characterized by high concentrations of RSS (up to 10 −3 M, mainly in the form of sulfide); nutrients (NH 4 +, up to 150 μM; PO 4 3−, up to 22 μM; SiO 4 4−, up to 400 μM) and DOC (up to 6 mg/l) as a result of the pronounced remineralization of allochthonous organic matter produced in the surface water. The depth position of anoxic water layer changes seasonally and is greatly influenced by rainfall, the influence of which is visible from decreased salinity in deeper layers as well. As a result of autumn/winter mixing, bottom water rich with nutrients is coming to the surface, supporting new phytoplankton and oxygen productions. Natural eutrophication of the lake is strongly influenced by nutrient recycling under anaerobic conditions. Turnover of lake water layers in September 1997 occurred so quickly that it resulted in the appearance of total anoxia and sulfide presence throughout the water column. The aerobic flora and fauna died and added to the oxygen demand and the production of nutrients. Those long-term datasets help in recognizing the general trend from unique cases, such as the anoxic event in 1997, which was related to anthropogenic eutrophication.

Anoxia Effects on CNS Function and Survival: Regional Differences

The effects of anoxia on function and survival of different central nervous system (CNS) areas were tested. As expected, synaptic function in a typical gray matter area of the brain, hippocampus, failed rapidly during 30 min of anoxia and did not recover. Mouse optic nerve and corpus callosum, two white matter (WM) areas of the brain, showed persistent function during total anoxia for periods as long as two hours. Moreover, even after two hours of anoxia followed by a recovery period, nearly half of the axons that were excitable at the outset remained functional. The corpus callosum contains a high percentage of unmyelinated axons while optic nerve axons are completely myelinated. These studies indicate that CNS structures vary greatly in their ability to function and survive anoxia. Mammalian WM, independent of myelination, is remarkably tolerant of anoxia implying that CNS axons generate enough ATP by anaerobic energy metabolism to sustain function.

Metabolic adaptation to prolonged anoxia in leaves of American cranberry (Vaccinium macrocarpon).

The indigenous North American Cranberry (Vaccinium macrocarpon), when cultivated in specially constructed cranberry bogs, is normally flooded in winter to prevent frost injury. This protection under ice can give rise to prolonged periods of anoxia, which depending on the state of the vines and environmental conditions, can cause severe oxygen-deprivation injury. An experimental study of the tolerance of cranberry vines to controlled total anoxia reveals that mature dark-green perennating leaves with high carbohydrate levels are able to survive prolonged periods of total oxygen-deprivation while younger newly formed leaves are readily damaged. During the anoxic treatment the mature leaves exhibit a marked downregulation of metabolism. Carbohydrate consumption and energy metabolism stabilize at low levels soon after the switch from aerobic to anaerobic pathways. Pathways such as TCA cycle or photosynthesis, which are non-operating during the anoxia treatment, are severely affected but still measurable after 28 days anoxia. In the post-anoxic period the perennating leaves rapidly re-establish their capacity for aerobic respiration and photosynthesis.

Effect of Anoxia on Respiration Rate (Fermentative Index) and Ethanol Production of Onion Bulbs (Allium cepa L.)

The physiological behavior, including carbon dioxide production, fermentative index (FI) and ethanolic production of onion bulbs kept under total anoxia (l00% N2) was investigated. During the first 24 hours, carbon dioxide production increased from 0.01 to 1.56 kPa Co2, and the average rate of the increase in CO2; production between 0 and 24 hours was 0.09 kPa/h. The Q10, of the fermentative index was l.9. Ethanol produced by onion bulbs kept under anoxia during 6 hours was temperature dependent, and was 0.563 and 0.760 pmol kg-1h-1 at 10 and 20°C respectively, while at 4°C the quantity produced was not detected. It is concluded that onion seems to be less tolerant to anoxia than other vegetables such as artichoke, cauliflower, tomato, potato and asparagus.

Long-term anoxia tolerance in leaves of Acorus calamus L. and Iris pseudacorus L.

Mature green leaves of Acorus calamus and Iris pseudacorus have been shown to survive at least 28 d of total anoxia in the dark during the growing season, increasing up to 75 d and 60 d in overwintering leaves in A. calamus and I. pseudacorus, respectively. During the period of anaerobic incubation the glycolytic rate is reduced, carbohydrate reserves are conserved and ethanol levels in the tissues reached an equilibrium. Prolonged anoxia significantly suppressed leaf capacity for respiration and photosynthesis. After 28 d of anoxia, respiratory capacity was reduced in A. calamus and I. pseudacorus by 80% and 90%, respectively. The photosynthetic capacity of leaves decreased by 83% in A. calamus and by 97% in I. pseudacorus after 28 d of anoxia. This reduction in photosynthetic capacity was accompanied by a modification of the chlorophyll fluorescence pattern indicating damage to the PSII reaction centre and subsequent electron transport. Chlorophyll content was only slightly reduced after 28 d under anoxia and darkness in A. calamus, whereas there was a 50% reduction in I. pseudacorus. On return to air A. calamus leaves that endured 28 d of anoxia recovered full photosynthetic activity within 7 d while those of I. pseudacorus had a lag phase of 3-10 d. This well-developed ability to endure prolonged periods of oxygen deprivation in both these species is associated with a down-regulation in metabolic activity in response to the imposition of anaerobiosis. It is suggested that when leaf damage eventually does take place in these species after protracted oxygen deprivation, it is anoxic rather than post-anoxic stress that is responsible.

Effects of hyperbaric oxygen on expression of fibrinolytic factors of human endothelium in a simulated ischaemia/reperfusion situation

Background : Treatment with hyperbaric oxygen (HBO 2 ) is controversial when treating disorders other than decompression sickness. Still, HBO 2 is a treatment modality that has gained recognition in certain situations of ischaemia reperfusion. However, not much is known about its effect on the endothelial cells. Based on earlier studies, the hypothesis was that HBO 2 treatment stimulates the release of fibrinolytic factors. The aim of the study was to investigate the effect of HBO 2 treatment on cultured endothelial cells in a simulated ischaemiareperfusion model. Methods : To mimic the clinical situation during ischaemia reperfusion, endothelial cells were subjected to anoxia for 8 h, followed by reperfusion with either HBO 2 or normobaric air for 1.5 h, and compared with an untreated control that was not exposed to anoxia. Components investigated were the fibrinolytic stimulator tissue plasminogen activator (t-PA), urokinase plasminogen activator (uPA) and the antagonist, plasminogen activator inhibitor type one (PAI-1). Results : Immediately after 8 h of total anoxia and reoxygenation with HBO 2 (for 1.5 h), the mean (SEM) concentrations of t-PA, PAI-1 and uPA were significantly increased compared to the other groups. The difference between the normobaric and control groups, measured at 1.5 h, 6 h and 24 h post-anoxia, persisted throughout the experiment. Conclusion : In this ischaemia-reperfusion model, HBO 2 stimulates the release of fibrinolytic factors. These observations might be relevant in trauma care in preventing thromboses and/or microembolization following ischaemia-reperfusion.

The calanoid copepod Acartia italica Steuer, phenomenon in the small saline Lake Rogoznica (Eastern Adriatic coast)

Lake Rogoznica, near Sibenik, Croatia, is a salt coastal lake with a maximum depth of about 15 m. During most of the year, this small, naturally eutrophied lake is highly stratified, with hypoxia/anoxia occurring in the bottom layer. Total anoxia, with the presence of hydrogen sulphide, was recorded on September 27, 1997. At that moment, massive mortality of all planktonic and benthic organisms was observed. After the anoxia, Acartia italica, the only planktonic copepod species of the lake, re-established quickly. Before anoxia, the population dynamics of the A.italica population depended primarily on predator/prey relationships. However, in the post-anoxic period, nutrient-phytoplankton-copepod relationships became more important, as there was no longer any predation pressure. From all accounts, it would appear that only A.italica is adapted to the extreme conditions which appear from time to time in the lake, and that this species has an important role in the functioning of the lake ecosystem. Acartia italica was very important for the gradual normalization of life in the lake after total anoxia.

Life, death and revival of the hypertrophic Mariager Fjord, Denmark

The bottom water in the Danish Mariager Fjord is normally anoxic but for 2 weeks in 1997, the water column became anoxic to the surface. The fjord is about 40 km long and mostly less than 2 km wide. The inner 17 km of the fjord is 10–30 m deep and the water in the deep basis at Mariager is nearly permanently anoxic below 15 m. The outer part consists of tidal plains with water depths around 1 m bordering a narrow, winding, 6-m deep channel held open by the tide. The Inner Fjord is presently hypertrophic with a primary production in the range of 800–900 g C m −2 year −1. Under normal environmental conditions, the mussel Mytilus edulis covers the slopes and banks between 1 and 8–10 m depths. In 1997, the summer in Denmark was unusually warm, sunny and calm. The wind induced very little mixing of the surface water and the input of oxygen from the air was therefore very limited. At some point, the limited oxygen input could not meet the mussels' and other organisms' oxygen demand. The Inner Fjord became anoxic and sulphidic to the very surface for a distance of 20 km. The aerobic flora and fauna died and added to the oxygen demand. The general conditions and special events resulting in the total anoxia are illustrated and discussed. The revival of the fjord ecosystem is commented upon, based on the first results of the ongoing investigations.

Spatial heterogeneity of the Tucuruí Reservoir (State of Pará, Amazonia, Brazil) and the distribution of zooplanktonic species.

With the purpose of analyzing the horizontal distribution of the zooplankton community of the Tucuruí Reservoir in the State of Pará, Brazil, collections were made at 16 stations during August 1988. The stations cover the regions called Caraipé (C), Araçagi (B) and Ararão (A), and represent the different compartments of this ecosystem in regard to the morphometry and the presence or absence of "flooded forest". Our findings showed the existence of three compartments with different limnological characteristics determined as a function of the system's morphometry, a factor that may have influenced the horizontal circulation of the reservoir's water and, consequently, led to physical, chemical and biological differences at each of the sampled stations. All the stations presented physical and chemical stratification and layers of total anoxia or reduced concentrations of oxygen dissolved at depths corresponding to the limit of the euphotic zone. With regard to the zooplankton, a differentiated distribution that was mainly quantitative was found among the compartments (density of organisms and proportion among the species) in the case of Cladocera and Copepoda, and basically qualitative in the case of Rotifera. On the other hand, the greatest densities of organisms for all the groups were recorded at the Caraipé stations. As for the spatial distribution, some species were more restricted or more abundant at given stations. Among the Cladocera, there was a greater abundance of Moina minuta, Bosmina hagmani and Bosminopsis deitersi at the Araçagi stations, while C. cornuta intermedia, C. cornuta rigaudi and C. cornuta cornuta were more plentiful in the Caraipé and Diaphanosoma birgei in the Ararão. Among the Rotifera, Trochosphaera aequatoriales and Lecane proiecta, Ascomorpha ecaudis, besides Polyartha cf vulgaris were restricted, respectively, to the Caraipé, Araçagi and Ararão stations, while the others, such as Keratella americana and Collotheca sp., were more broadly distributed. As regards Copepoda Calanoida, the dominating species was found to be Notodiaptomus maracaibenses, followed by Notodiaptomus henseni and Argyrodiaptomus azevedoi. The most abundant Cyclopoda species was Thermocyclops minutus, although Thermocyclops decipiens, Mesocyclops longisetus, Mesocyclops meridianus e Metacyclops sp. were also found.

Anaerobic processes in activated sludge

We found anoxic zones in aerated activated sludge flocs, and demonstrated denitrification under normal operating conditions. Sulfate reduction was not found. Micro-environments and microbial conversions in flocs from bulking and non-bulking activated sludge were determined with microsensors for H2S, O2, NO2− and NO3−. Denitrification and sulfate reduction rates were mmeasured with 15N- and 35S-tracer techniques. We showed that under normal reactor conditions (ca. 20% air saturation) anoxic zones develop within flocs allowing denitrification. The denitrification rates amounted to 40% of the rates under anoxic conditions. At 100% air saturation no anoxic zones were found and no denitrification occurred. However, in flocs from bulking sludge (at 20% air saturation) anoxic zones were absent and denitrification did not occur. In bulking sludge only at total anoxia was denitrification found. Confocal microscopy showed that flocs from bulking sludge were much looser than those from non-bulking sludge. The absence of anoxic zones and of denitrification was attributed to the open floc structure, allowing advective oxygen transport. Sulfate reduction was not detected in any of the sludges tested by microsensors or by tracer techniques even under anoxic conditions. this indicates that the sulfur cycle (sulfate reduction and sulfide oxidation) does not play a role in mineralization processes and bulking in activated sludge. Preliminary molecular work (in situ hybridization with the 16S-rRNA probe SRB385) indicated the presence of small amounts of sulfate reducing bacteria in all sludges. Either the probe is not specific or the sulfate reducers present are not active under reactor conditions.

Anaerobic processes in activated sludge

We found anoxic zones in aerated activated sludge flocs, and demonstrated denitrification under normal operating conditions. Sulfate reduction was not found. Micro-environments and microbial conversions in flocs from bulking and non-bulking activated sludge were determined with microsensors for H2S, O2, NO2− and NO3−. Denitriftcation and sulfate reduction rates were measured with 15N- and 35S-tracer techniques. We showed that under normal reactor conditions (ca. 20% air saturation) anoxic zones develop within flocs allowing denitrification. The denitrtftcation rates amounted to 40% of the rates under anoxic conditions. At 100% air saturation no anoxic zones were found and no denitrification occurred. However, in flocs from bulking sludge (at 20% air saturation) anoxic zones were absent and denitrification did not occur. In bulking sludge only at total anoxia was denitrification found. Confocal microscopy showed that flocs from bulking sludge were much looser than those from non-bulking sludge. The absence of anoxic zones and of denitrification was attributed to the open floc structure, allowing advective oxygen transport.Sulfate reduction was not detected in any of the sludges tested by microsensors or by tracer techniques even under anoxic conditions. This indicates that the sulfur cycle (sulfate reduction and sulfide oxidation) does not play a role in mineralization processes and bulking in activated sludge. Preliminary molecular work (in situ hybridization with the 16S-rRNA probe SRB385) indicated the presence of small amounts of sulfate reducing bacteria in all sludges. Either the probe is not specific or the sulfate reducers present are not active under reactor conditions.

3 Metabolic acidosis and fetal reserve

Dr Bailey, Director of the National Institute of Neurological Diseases and Blindness, Bethesda, Maryland, introduced the 1956 symposium on 'Neurological and psychological deficits of asphyxia neonatorum' by saying. 'Medical research in regard to cerebral palsy and other neurological disabilities has been relatively neglected. For a truly comprehensive attack on this problem we must focus a sharper scientific search for greater knowledge of those adverse biological factors which operate in the perinatal period. The proper point of departure in such a search is through controlled animal observations, for which purpose monkeys are best suited' (Windle, 1958). Our understanding of the significance of asphyxia is built on the foundation of the laboratory research that has followed upon this initiative. Laboratory studies in fetal monkeys and fetal lambs have clearly demonstrated that the fetus can initially compensate for an asphyxial insult and protect the vital organs. However, if the hypoxaemia progresses to a severe metabolic acidosis and cardiovascular decompensation with hypotension, brain damage will occur. There is a growing body of clinical evidence that supports the contention that the human fetus responds in a similar manner. The varied nature of hypoxic and ischaemic insults has been well demonstrated in animal studies. Total anoxia and isolated cerebral ischaemia are uncommon events in the human fetus. The common insult, particularly during labour, is a degree of hypoxia present over a variable period of time. This is fortunate in that such insults are associated with a period of fetal compensation that may last several hours, during which time a diagnosis of a developing metabolic acidosis can be made. This is the clinician's window of opportunity, when a definitive diagnosis of an asphyxial insult can be made and if necessary intervention made before the threshold of decompensation has been reached. A consensus on the threshold of decompensation has yet to be achieved. However, there is a growing body of evidence that the threshold is in the range of an umbilical artery base deficit of 12-16 mmol/l. Since the aim of the obstetrician during labour is the prevention of asphyxial morbidity and mortality, the determination of this threshold is important to provide criteria for clinical action. A blood gas and acid-base assessment with the determination of a metabolic acidosis is the best measure of an asphyxial insult that may be of clinical significance. The definition of the threshold of metabolic acidosis requiring intervention will continue to be clarified with future clinical research. However, there are many factors that will influence the fetal response to an asphyxial insult, which may require that a range of threshold be acknowledged. The effect upon the fetus will be influenced by whether the asphyxial event is the first or the last of a series of asphyxial episodes, and the duration of the asphyxial episode. The characteristics of the fetus, i.e. maturity (pre-term versus term), and fetal growth small for gestational age (SGA) or appropriate for gestational age (AGA) may influence the fetal response to an asphyxial insult. Improved understanding of these issues will provide a better rationale for clinical management.