Evaluation of oxygen transfer efficiency under process conditions using the dynamic off-gas method.

The off-gas method can be used to investigate standard oxygen transfer efficiencies under process conditions (alphaSOTE) over the operating life of an aeration system. A method to evaluate alphaSOTE is described in detail by US and German standards. The standards, however, do not describe how to evaluate dynamic changes in aSOTE over a day, which can be useful to uncover problems and unfavourable process conditions. Based on over three years experience gained in off-gas testing in Berlin wastewater treatment plants (WWTPs) under operating conditions, a method to evaluate and interpret the dynamic changes in oxygen transfer is presented. The application of the dynamic off-gas method brings important additional information, which can be used to increase operational efficiency of the aeration basin and to increase process reliability, with a relatively small increase in effort. This paper shows how to perform dynamic measurements under process conditions. Some results of such measurements under dynamic process conditions, performed in a Berlin WWTP, are discussed.

Evaluation of ceramic and membrane diffusers under operating conditions with the dynamic offgas method.

The aeration systems of two full-scale, activated-sludge basins were compared during a period of three years, under the same operating conditions, using dynamic offgas testing. Only the material of the diffuser was different (membrane versus ceramic-tube diffusers). The investigation has shown that, although the membrane diffusers have higher initial standard-oxygen-transfer efficiency (alphaSOTE) and standard-aeration efficiency (alphaSAE), these decreased over time, while the alphaSAE of the ceramic diffusers started lower, but increased slightly over the whole period. A cost comparison makes clear how important it is to evaluate the aeration system under process conditions. The operating costs were the dominant factor (approximately 10x higher than capital costs), and operating costs were approximately 20% higher for membrane versus ceramic diffusers. The poor performance of the membrane-tube diffusers under process conditions could be explained on the basis of the actual alphaAE values in the basin, not the standardized values.

Potential of OUR and OTR measurements for identification of activated sludge removal processes in aerated basins.

In order to develop a process control scheme to reduce energy costs for aeration in activated sludge systems with biological P removal, pre-denitrification and nitrification stages, the spatial distribution of carbon oxidation and nitrification was evaluated over a long full-scale plug flow aeration basin using an externally measured specific oxygen uptake rate (sOUR) and in basin measurement of the actual specific oxygen transfer rate (sOTR) with off-gas testing as well as with the calculated oxygen demand from NH4-N concentrations (sOTR(N)). Using a simple static model, a gas phase balance on oxygen and carbon dioxide, sOTR(N) values were also calculated from off-gas testing. Comparison of sOTR(N) to sOTR and sOUR for carbon oxidation (sOUR(C)) to nitrification (sOUR(N)) at different loading conditions allowed the oxidation processes to be followed over the three zones of the aeration basin. As expected, the distribution depended on the dissolved oxygen concentration (DO) in the basin. However, the major change was in the C-oxidation rate and not the nitrification rate. At a low DO, and when NH4-N was present in the zone, the amount of oxygen transferred for nitrification was nearly the same, but the overall sOTR was lower. The externally measured sOUR was only useful when it was differentiated into sOUR(N) and sOUR(C). sOUR(N) could be used to predict the nitrification rate in the basin. With further refinement, the gas phase balance model has potential to be used to monitor the degree of nitrification over the basin length. This can be integrated into a control scheme to reduce aeration costs by adjusting the DO setpoint according to loading conditions in the

A control strategy for reducing aeration costs during low loading periods.

The efficiency of the aeration system in a full-scale activated sludge basin with 3 separately controlled aeration zones was improved for the low loading period in summer. The air flow rate to each aeration zone is currently regulated to hold a preset dissolved oxygen concentration (DO). Four different DO setpoint combinations were tested, each one for a one week period, using dynamic off-gas testing to measure the standardised oxygen transfer efficiency (alphaSOTE). As the DO setpoints were lowered, the total air flow rate to the basin decreased initially. A low DO in the first zones slowed biomass activity and pushed the load towards the end of the aeration basin. The relationship between alphaSOTE and the specific diffuser flow rate qD is different for each zone. In Zone 1 there was a strong decrease in alphaSOTE as qD increased, while Zones 2 and 3 were fairly independent of qD, Zone 2 at a higher level than Zone 3. Aeration costs were reduced by 15% for the most efficient combination. To achieve even more savings, a control strategy adjusting oxygen transfer rates over the aeration basin to the necessary oxygen transfer rates is suggested. It is based on changing the DO setpoints to reach the lowest total air flow rate while meeting the effluent requirements.

Comparison of the efficiency of large-scale ceramic and membrane aeration systems with the dynamic off-gas method.

The aeration systems of two full-scale activated sludge basins were compared over 2.5 years under the same operating conditions using dynamic off-gas testing. Only the material of the diffuser was different, membrane vs. ceramic tube diffusers. The experimental design took the complexity and dynamics of the system into consideration. The investigation has shown that, although the membrane diffusers have higher initial standard oxygen transfer efficiency (SOTE) and standard aeration efficiency (SAE), these decreased over time, while the SAE of the ceramic diffusers started lower, but increased slightly over the whole period. Measurement of air distribution in the basins along with dissolved oxygen concentration profiles have provided important information on improving process control and reducing energy costs. The results show that dynamic off-gas testing can effectively be used for monitoring the aeration system and to check design assumptions under operating conditions. The information can be used to improve the design of new aeration systems or in retro-fitting existing basins.

BMPs as mediators of roof plate repulsion of commissural neurons.

During spinal cord development, commissural (C) neurons, located near the dorsal midline, send axons ventrally and across the floor plate (FP). The trajectory of these axons toward the FP is guided in part by netrins. The mechanisms that guide the early phase of C axon extension, however, have not been resolved. We show that the roof plate (RP) expresses a diffusible activity that repels C axons and orients their growth within the dorsal spinal cord. Bone morphogenetic proteins (BMPs) appear to act as RP-derived chemorepellents that guide the early trajectory of the axons of C neurons in the developing spinal cord: BMP7 mimics the RP repellent activity for C axons in vitro, can act directly to collapse C growth cones, and appears to serve an essential function in RP repulsion of C axons.

RET proto-oncogene is important for the development of respiratory CO2 sensitivity.

Brain stem muscarinic cholinergic pathways are important in respiratory carbon dioxide (CO2) chemosensitivity. Defects in the muscarinic system have been reported in children with congenital/developmental disorders of respiratory control such as sudden infant death syndrome (SIDS) and congenital central hypoventilation syndrome (CCHS). This early onset of disease suggests a possible genetic basis. The muscarinic system is part of the autonomic nervous system which develops from the neural crest. Ret proto-oncogene is important for this development. Thus, a potential role for ret in the development of respiratory CO2 chemosensitivity was considered. Using plethysmography, we assessed the ventilatory response to inhaled CO2 in the unanesthetized offsprings of ret +/- mice. Fractional increases in minute ventilation during hypercapnia relative to isocapnia were 5.1 +/- 3.2, 3.0 +/- 1.6 and 1.4 +/- 0.8 for the ret +/+, ret +/- and ret +/- mice, respectively. The ret knockout mice have a depressed ventilatory response to inhaled CO2. Therefore, the ret gene is an important factor in the pathway of neuronal development which allow respiratory CO2 chemosensitivity.

Increased expression of laminin-1 and collagen (IV) subunits in the aganglionic bowel of ls/ls, but not c-ret -/- mice.

Extracellular matrix molecules, including laminin, affect the development of enteric neurons and accumulate in the aganglionic colon of ls/ls mice. Quantitative Northern analysis revealed that mRNAs encoding the beta 1 and gamma 1 subunits of laminin and collagens alpha 1(IV) and alpha 2(IV) are increased in the colons of ls/ls mice. Transcripts of laminin alpha 1 were evaluated quantitatively with reverse transcription and the competitive polymerase chain reaction (RT-cPCR). The abundance of laminin alpha 1 transcripts was developmentally regulated, but greater in the ls/ls than the wild-type colon at each age examined. In situ hybridization revealed that transcripts in the colon encoding laminin alpha 1 and beta 1 and collagen alpha 2(IV) were initially expressed in the endoderm, but by E15, expression shifted to cells of the colonic mesenchyme (ls/ls > wild type) where crest-derived cells migrate. The expression of laminin alpha 1 was examined in the totally aganglionic intestine of E15 and newborn c-ret -/- mice, to determine whether an increase occurs when neurogenesis fails independently of the ls/ls defect. RT-cPCR revealed no difference from control in mRNA encoding laminin alpha 1 in the c-ret -/- colon in either E15 or newborn animals. The accumulation of immunohistochemically demonstrable laminin that is prominent in the newborn ls/ls colon could not be detected in that of c-ret -/- animals. These observations suggest that transcripts encoding laminin-1 and collagen (IV) are increased in the colon and surrounding pelvic mesenchyme of ls/ls mice because of an intrinsic lesion, rather than a secondary consequence of aganglionosis. The data are compatible with the hypothesis that the increased expression of laminin-1 contributes to the failure of crest-derived cells to complete their colonization of the ls/ls colon.

Renal agenesis and hypodysplasia in ret-k- mutant mice result from defects in ureteric bud development.

The c-ret gene encodes a receptor tyrosine kinase that is expressed in the Wolffian duct and ureteric bud of the developing excretory system. Newborn mice homozygous for a mutation in c-ret displayed renal agenesis or severe hypodysplasia, suggesting a critical role for this gene in metanephric kidney development. To investigate the embryological basis of these defects, we characterized the early development of the excretory system in mutant homozygotes, and observed a range of defects in the formation, growth and branching of the ureteric bud, which account for the spectrum of renal defects seen at birth. Co-culture of isolated ureteric buds and metanephric mesenchyme show that the primary defect is intrinsic to the ureteric bud. While the mutant bud failed to respond to induction by wild-type mesenchyme, mutant mesenchyme was competent to induce the growth and branching of the wild-type bud. Furthermore, the mutant metanephric mesenchyme displayed a normal capacity to differentiate into nephric tubules when co-cultured with embryonic spinal cord. These findings suggest a model in which c-ret encodes the receptor for a (yet to be identified) factor produced by the metanephric mesenchyme, which mediates the inductive effects of this tissue upon the ureteric bud. This factor appears to stimulate the initial evagination of the ureteric bud from the Wolffian duct, as well as its subsequent growth and branching.

Common origin and developmental dependence on c-ret of subsets of enteric and sympathetic neuroblasts.

c-ret encodes a tyrosine kinase receptor that is necessary for normal development of the mammalian enteric nervous system. Germline mutations in c-ret lead to congenital megacolon in humans, while a loss-of-function allele (ret.k-) causes intestinal aganglionosis in mice. Here we examine in detail the function of c-ret during neurogenesis, as well as the lineage relationships among cell populations in the enteric nervous system and the sympathetic nervous system that are dependent on c-ret function. We report that, while the intestine of newborn ret.k- mice is devoid of enteric ganglia, the esophagus and stomach are only partially affected; furthermore, the superior cervical ganglion is absent, while more posterior sympathetic ganglia and the adrenal medulla are unaffected. Analysis of mutant embryos shows that the superior cervical ganglion anlage is present at E10.5, but absent by E12.5, suggesting that c-ret is required for the survival or proliferation of sympathetic neuroblasts. In situ hybridization studies, as well as direct labelling of cells with DiI, indicate that a common pool of neural crest cells derived from the postotic hindbrain normally gives rise to most of the enteric nervous system and the superior cervical ganglion, and is uniquely dependent on c-ret function for normal development. We term this the sympathoenteric lineage. In contrast, a distinct sympathoadrenal lineage derived from trunk neural crest forms the more posterior sympathetic ganglia, and also contributes to the foregut enteric nervous system. Overall, our studies reveal previously unknown complexities of cell lineage and genetic control mechanisms in the developing mammalian peripheral nervous system.

RET-deficient mice: an animal model for Hirschsprung's disease and renal agenesis.

Receptor tyrosine kinases play a critical role in transducing signals involved in cell growth and differentiation. The c-ret proto-oncogene is a member of the receptor tyrosine kinase gene superfamily originally identified by its transforming ability. Somatic mutations of c-ret are responsible for a large proportion of thyroid papillary carcinomas, while germ-line mutations are responsible for multiple endocrine neoplasia types 2A and 2B, dominantly inherited cancer syndromes characterized by multiple tumours of neuroectodermal origin. In addition to its role in tumour formation. c-ret is thought to have a developmental role since mutations of the gene have been implicated in the aetiology of Hirschsprung's syndrome (congenital megacolon). A targeted mutation in the murine c-ret locus shows that the ret receptor is required for normal development of two lineally unrelated systems, the excretory system and the enteric nervous system.

Isolation and characterization of a chicken homolog of the c-ret proto-oncogene.

The c-ret proto-oncogene encodes a receptor tyrosine kinase that plays important roles in human disease and in normal mammalian development. Mutations in the human RET gene are associated with multiple endocrine neoplasia syndromes and Hirschsprung's disease in humans, while targeted mutagenesis of murine c-ret resulted in severe developmental abnormalities affecting the excretory and peripheral nervous systems. To examine the evolutionary conservation of the ret protein sequence and its developmental expression pattern, we isolated and sequenced cDNA clones of chicken c-ret and examined its expression in chick embryos and adult tissues. The cytoplasmic domains of chicken and human ret were relatively well conserved (91% similar), but the extracellular domains were more divergent (68% similar), although the conservation of cysteine residues in this region suggests a conserved secondary structure. As in mouse and human, chicken c-ret encodes two protein isoforms. The number and sizes of the transcripts were similar to those in human and mouse cells, and during chick embryogenesis, c-ret mRNA was observed in many of the same sites as in the mouse, including the Wolffian duct and ureteric bud, the enteric, dorsal root, sympathetic and facioacoustic ganglia, and the ventral spinal cord. Evolutionary differences in expression were observed in the trigeminal ganglion, the ventral roots of the spinal cord, the mesenchymal cells of the branchial arches and the adult testes. The results are discussed with regard to the role of the ret receptor in normal development and disease.

Defects in the kidney and enteric nervous system of mice lacking the tyrosine kinase receptor Ret.

Receptor tyrosine kinases (RTKs) are cell-surface molecules that transduce signals for cell growth and differentiation. The RTK encoded by the c-ret proto-oncogene is rearranged and constitutively activated in a large proportion of thyroid papillary carcinomas, and germ-line point mutations in c-ret seem to be responsible for the dominantly inherited cancer syndromes multiple endocrine neoplasia (MEN) types 2A and B. The gene is expressed in the developing central and peripheral nervous systems (sensory, autonomic and enteric ganglia) and the excretory system (Wolffian duct and ureteric bud epithelium) of mice, indicating that it may play a role in normal development. Here we show that mice homozygous for a targeted mutation in c-ret develop to term, but die soon after birth, showing renal agenesis or severe dysgenesis, and lacking enteric neurons throughout the digestive tract. Ret is thus an essential component of a signalling pathway required for renal organogenesis and enteric neurogenesis.