Aerobic/anoxic post-treatment of anaerobically digested sewage sludge as an alternative to biological nitrogen removal from reject water.

Stabilisation and biological nitrogen removal (BNR) of anaerobically digested sewage sludge were studied in a post-aeration reactor at pilot scale working under alternating anoxic-aerobic conditions. Digested sludge came from a two-stage anaerobic digestion (thermophilic + mesophilic). The best post-aerator performance was achieved when working at an HRT of 10 days (4 days aerobic; dissolved oxygen of 1.8 mg L(-1)) and VS content in the feed no lower than 6.7 g L(-1). Free ammonia concentration values in the effluent above 1.5 mg N L(-1) (around 150 mg NH4 (+)-N L(-1) at pH 7) were necessary to promote the BNR over nitrite. Removal efficiencies up to 80 % NH4 (+)-N, 50-55 % total nitrogen and 15-20 % VS were recorded in this study, with no external addition of chemicals. A nitrogen mass balance revealed that the high percent of NH4 (+)-N assimilated in heterotrophic growth was counteracted with that released in ammonification and fermentation, leading to a NH4 (+)-N removal mainly related to biological nitritation/denitritation.

Model-based operational guidelines of a bioprocess for biological nitrogen removal and complete stabilisation of anaerobically digested sewage sludge.

The concept of one-stage reactor system for biological nitrogen removal over nitrite of ammonium high loaded sidestreams is going to be applied to remove nitrogen from anaerobically digested sewage sludge and to achieve its complete stabilisation. Dealing with sludge, the organic matter needed to denitrify is present in the inflow as particulate substrate, which requires a hydrolysis step. The latter implies high anoxic hydraulic retention time (HRT). During both aerobic and anoxic phases, ammonium is released which implies the need to enlarge aerobic HRT. Both effects lead to a total HRT higher than those for nitrification-denitrification of wastewater with soluble substrate. The purpose of this paper is to define, by computer simulation, a set of theoretical criteria, which will be applied later to the operation of a pilot-scale post-aeration reactor to be located in a Spanish WWTP. These criteria will be defined by simulating the reactor performance under different operating conditions. As a conclusion, some operation guidelines have been established for the above-mentioned scenario in terms of aerobic and anoxic retention time, dissolved oxygen concentration and effluent requirements (NH(4)(+), NO(2)(-) and NO(3)(-)).

A multivariate Poisson regression model for count data.

We propose a new technique for the study of multivariate count data. The proposed model is applied to the study of the number of individuals several fossil species found in a set of geographical observation points. First, we are proposing a multivariate model based on the Poisson distributions, which allows positive and negative correlations between the components. We are extending the log-linear Poisson model in the multivariate case through the conditional distributions. For this model, we obtain the maximum likelihood estimates and compute several goodness of fit statistics. Finally we illustrate the application of the proposed method over data sets: various simulated data sets and a count data set of various fossil species.

Integration of equalisation tanks within control strategies for anaerobic reactors. Validation based on ADM1 simulations.

The combination of equalisation tanks and anaerobic digesters represents a typical design scenario within the treatment of industrial wastewaters. In this context, if the hydraulic capacity of the equalisation tanks is effectively handled, significant improvements in the performance of anaerobic digesters can be achieved in terms of process stability and biogas production. This paper presents a rule-based control strategy for anaerobic reactors with the objective of maximising in the long-term the net production of biogas. The control algorithm combines real-time information about the state of the anaerobic digester with on-line measurements about the wastewater volume of the equalisation tank in order to set permanently the appropriate production of biogas. Such a strategy guarantees a continuous influent flow so that emptying and overflowing episodes in the equalisation tank can be prevented. Aiming at a further full-scale implementation, only reliable and cost-effective on-line instrumentation has been considered within the control architecture. The performance of the proposed control approach has been validated for an anaerobic hybrid configuration (AHR) by simulation using the IWA ADM1 model.

Mathematical modelling of autothermal thermophilic aerobic digesters.

This paper presents a new mathematical model for Autothermal Thermophilic Aerobic Digesters. The reactor has been modelled as two completely mixed volumes to separately predict the behaviour of the liquid and gaseous phases as well as the interrelation between them. The model includes biochemical transformations based on the standard Activated Sludge Models of IWA, as well as physico-chemical transformations associated with the chemical equilibria and the mass transfer between the liquid and the gaseous phases similar to those proposed in the ADM1 of IWA. An energy balance has also been included in the model in order to predict the temperature of the system. This thermal balance takes into account all those biochemical and physico-chemical transformations that entail the most relevant heat interchanges. Reactor performance has been explored by simulation in two different scenarios: in the first where it acts as the initial stage in a Dual system, and in the second where it acts as a single-stage treatment. Each scenario enabled the identification of the relevance of the different parameters.

Algebraic solution of the mass balanced ADM1 to predict the steady state and to optimise the design of the anaerobic digestion of sewage sludge.

This paper proposes an algebraic solution of the mass and charge balanced ADM1 model to predict the steady state performance of an anaerobic digester for sewage sludge treatment. The algebraic solution consists of three sequential stages: a kinetic stage that considers only the slowest transformations of the model, a stoichiometric stage based on the complete mass fluxes of the biological process and a physicochemical stage from which some digester outputs are calculated. The predictive capacity and the applicability of this model solution are corroborated by its comparison to the differential equation's model solution and the experimental data of a real case study. The algebraic solution is used to explore the digester response under different operational conditions. An example of application is used to verify the potential of the algebraic solution to be used, together with optimisation algorithms, for optimising the design of the digester and the operational conditions for specified performance criteria, such as effluent quality.

Mathematical modelling of the anaerobic hybrid reactor.

This paper presents a new mathematical model for the anaerobic hybrid reactor (AHR) (a UASB reactor and an anaerobic filter in series) and its experimental calibration and verification. The model includes a biochemical part and a mass transport one, which considers the AHR as two contact reactors in series. The anaerobic process transformations are described by the model developed by Siegrist et al. The fraction (F) of solids in the clarification zone of the UASB reactor that leaves this first reactor is the key physical parameter to be estimated. The main parameters of the model were calibrated using experimental results from a bench-scale AHR fed with real slaughterhouse wastewater. The fraction of inert particulate COD in the influent and the factor F were estimated by a trial and error procedure comparing experimental and simulated results of the mass of solids in the lower tank and the VSS concentration in the AHR effluent. A good fit was obtained. The final verification was carried out by comparing a set of experiments with simulated data. The model's capability to predict the process performance was thus proved.

ADM1-based methodology for the characterisation of the influent sludge in anaerobic reactors.

This paper presents a systematic methodology to characterise the influent sludge in terms of the ADM1 components from the experimental measurements traditionally used in wastewater engineering. For this purpose, a complete characterisation of the model components in their elemental mass fractions and charge has been used, making a rigorous mass balance for all the process transformations and enabling the future connection with other unit-process models. It also makes possible the application of mathematical algorithms for the optimal characterisation of several components poorly defined in the ADM1 report. Additionally, decay and disintegration have been necessarily uncoupled so that the decay proceeds directly to hydrolysis instead of producing intermediate composites. The proposed methodology has been applied to the particular experimental work of a pilot-scale CSTR treating real sewage sludge, a mixture of primary and secondary sludge. The results obtained have shown a good characterisation of the influent reflected in good model predictions. However, its limitations for an appropriate prediction of alkalinity and carbon percentages in biogas suggest the convenience of including the elemental characterisation of the process in terms of carbon in the analytical program.

Mass and charge conservation check in dynamic models: application to the new ADM1 model.

This paper proposes a systematic methodology for the analysis of the mass and charge balances in dynamic models expressed using the Petersen matrix notation. This methodology is based on the definition of the model components via elemental mass fractions and in the estimation of the COD as a function of the redox equations associated with these elements. This approach makes the automatic calculation of all the stoichiometric coefficients under different measuring units and the study of COD, charge or mass fluxes easier. As an example of its application this methodology was applied to the ADM1 in order to illustrate its usefulness for the analysis of organic matter characterisation, nitrogen release or biogas composition in anaerobic digestion. The application of the methodology for a rigorous integration of different IWA models is proposed for further study.

Two hereditary defects related to vitamin D metabolism map to the same region of human chromosome 12q13-14.

We have localized the locus for the vitamin D receptor (VDR) responsible for hypocalcemic vitamin D-resistant rickets (HVDRR), close to the pseudovitamin D-deficient rickets (PDDR) locus, another disorder related to impaired vitamin D metabolism. PDDR (formerly vitamin D dependency type I, VDD1) was recently mapped to human chromosome 12q14 by linkage analysis. Here we report on the assignment of VDR to 12q13-14 by in situ hybridization and by linkage analysis. Linkage analysis between VDR, PDDR, and several RFLP markers show close linkage, with no recombination (theta = 0) between VDR and PDDR (Z = 1.94), a COL2A1 haplotype (Z = 4.03), ELA1 (Z = 0.98), and D12S15 (Z = 4.17). The analysis of extended haplotypes in one of the PDDR families provides evidence for recombination between VDR and PDDR and localizes VDR together with COL2A1 proximal to PDDR. Complete allelic association detected between VDR and COL2A1 loci on PDDR chromosomes and lower association between VDR and PDDR suggests a VDR location very close to COL2A1 and one more distant to PDDR. We propose the following order of loci: (VDR, COL2A1), (PDDR, ELA1, D12S15), D12S4, (D12S14, D12S17), D12S6. Thus, two clearly distinct loci involved in the control of vitamin D activity map close to each other in the region 12q13-14.

Molecular analysis in newborns from Texas affected with galactosemia.

The spectrum of mutations in the Galactose-1-phosphate uridyl transferase (GALT) gene is described in 11 cases of classic galactosemia and 38 of Duarte-2 type identified by the Texas Newborn Screening Program. Blinded studies were done by automated DNA sequencing of all the 11 exons and the exon-intron boundaries of the GALT gene using genomic DNA isolated from dry blood spots. Fourteen different mutations (11 missense mutations, 2 nonsense mutations and 1 splicing mutation) were detected in 94 of the 98 mutant alleles (diagnostic efficiency of 96%). The prevalent mutations were N314D (41%), Q188R (37%) and K285N (4%). The other less frequent mutations were IVS2-2A>G and S135L (3% each), T138M (2%) and T23A, H184Q, Y251S, L195P, Q207X, L264X, Q344K, and A345D (1% each). Three novel mutations, T23A, Q207X, and A345D, were identified. Our study supports previous findings that N314D and Q188R are prevalent in Hispanics and Whites and K285N was only observed in Whites. The IVS2-2A->G mutation is probably ethnic specific because it was identified exclusively in Hispanics. S135L, a prevalent mutation in Blacks, was also present in 3 Hispanics. Two unusual genotypes were observed in 2 patients homozygous for the Duarte-2 N314D allele and heterozygous for a novel mutation (Q207X- N314D/N314D in a classic galactosemia and T23A- N314D/N314D in a Duarte-2 case). The detection of GALT gene mutations in newborns from Texas should focus first on N314D, Q188R, K285N, IVS2-2A>G, S135L and T138M. Other exons and exon-intron boundaries would have to be studied if either one or no mutations are found in the primary screening.

Molecular analysis of phenylketonuria (PKU) in newborns from Texas.

This study describes the mutations at the phenylalanine hydroxylase (PAH) locus in patients with the diagnosis of classic PKU (n=18), hyperphenylalaninemia (HPA) variant (n=9) and benign persistent hyperphenylalaninemia (HPA) (n=13) who were identified by the Texas Newborn Screening Program. Blinded studies were done by sequencing of the 13 exons and exon-intron boundaries of the PAH gene in genomic DNA isolated from dry blood spots. Thirty-six different mutations, including 25 missense mutations, six splice mutations, three deletions and two nonsense mutations were detected in 75 of the 80 mutant alleles (94%). The prevalent mutations were R408W (19%), V388M and IVS10nt-11g->a (6% each), Y414C (5%) and H170D, A403V, T380M and IVS7nt1g->a (4% each). Two novel missense mutations were identified in exon 5 (H170D and N167S). There was genotype/phenotype correlation in 33/40 cases (83%). For this population, exons 12, 11, 7, 5 and 8, which carry 78% of the mutations, would have to be screened first. However, the other exons must be studied when either one or no mutations are found in the primary screening. Hum Mutat 17:523, 2001.

Maternal complex chromosome rearrangement ascertained through a del (13)(q12.1q14.1) detected in her mildly affected daughter.

A maternal complex chromosome rearrangement (CCR) involving chromosomes 2, 13, and 20 was ascertained in a normal female through the diagnosis of a deletion of 13q in her daughter. The child has mild clinical features and developmental delay consistent with proximal deletions of 13q that do not extend into band q32 and a del(13)(q12q14.1) that does not involve the retinoblastoma locus by FISH. Maternal studies by GTG banding and FISH showed a complex karyotype with bands 13q12.3-->13q12.1::20p13 translocated to 2p13 and bands 2pter-->2p13::13q12.3-->13q14.1 translocated into band 20p13. This would be the first report of an interstitial deletion of 13q inherited from a parental complex chromosome rearrangement.

"De novo" duplication Xq23-->Xq26 of paternal origin in a girl with a mildly affected phenotype.

We report a de novo dup(X)(q23-->q26) in a 3-year-old girl with growth retardation, developmental delay, and minor anomalies. X-inactivation in lymphocytes by BRDU labeling showed the abnormal X was late replicating. The androgen receptor assay (HAR) demonstrated a skewed methylation (88.8%) of the paternal allele and a 11.2% methylation of the maternal allele. These data, which suggest the duplication was paternally inherited, are the first parental-origin identification of a duplication Xq. The mild phenotype of the patient may be related to the size and region of the duplication, the low percentage of a dup(X) active detected by the HAR assay, or a combination of these mechanisms.

Constitutional del(19)(q12q13.1) in a three-year-old girl with severe phenotypic abnormalities affecting multiple organ systems.

We present the clinical, cytogenetic, and molecular studies on a constitutional deletion of 19q ascertained prenatally due to decreased fetal activity and IUGR. Chromosome analysis by GTG banding on amniocytes suggested a del(19)(q13.1q13.3), but the analysis of microsatellites by PCR demonstrated that the deletion involved the distal segment of q12 and the proximal segment of q13.1 (15 cM). The severely affected female infant born at 38 weeks has clinical findings that may be related to haploinsufficiency of specific genes within 19q12.1-->q13.1 that control important processes of normal development and cell function.

Mild phenotypic effects of a de novo deletion Xpter-->Xp22.3 and duplication 3pter-->3p23.

We report on a girl with a de novo monosomy Xpter-->Xp22.3 and trisomy 3pter-->3p23, normal development and stature, mildly affected phenotype, and learning disabilities with a low normal level of intelligence. Late replication studies using BudR demonstrated that the entire der(X) was inactive in 30% of cells. In 62% of cells the inactivation did not spread to the autosomal segment in the der(X). The normal X was inactivated in 8% of cells. Quantitative X-inactivation studies using the human androgen receptor locus assay (HAR) on peripheral leukocytes and buccal epithelial cells showed extreme skewing of methylation (90.4% of the paternal allele). The correlation of cytogenetic and molecular data suggest that the mild phenotype of the proposita is most likely due to preferential inactivation of the entire der(X), which seems to be of paternal origin.

De novo partial duplications 1p: report of two new cases and review.

We describe two de novo intrachromosomal duplications of 1p. One case is a dir ins dup(1)(q21p21p31) in a newborn girl with low birth weight, growth retardation, and tetralogy of Fallot. The other is a 10-month-old girl with developmental delay, craniosynostosis, plagiocephaly, and an inv dup 1p34.1p31. Although, these patients have manifestations in common with previous cases, they do not establish a syndrome. Interestingly, all males with duplications spanning 1p31 had genital anomalies, whereas females with duplications of the same region had normal genitalia. Thus, genes within 1p31 appear to control the development of male genitalia and tentatively exclude effects of tda1, a sex-determining gene in a region of mouse chromosome 4 syntenic to 1p36 in man. However, it is necessary to identify the human tda1 homologue and candidate genes within 1p31 before drawing final conclusions.

Reverse dot-blot hybridization as an improved tool for the molecular diagnosis of point mutations in congenital adrenal hyperplasia caused by 21-hydroxylase deficiency.

BACKGROUND: More than 90% of cases of congenital adrenal hyperplasia (CAH) are caused by mutations of the CYP21 gene that result in deficiencies of the enzyme 21-hydroxylase. Allele-specific PCR, allele-specific oligonucleotide hybridization, and Southern blot analysis are the most common methods to detect point mutations and deletions in the CYP21 gene. METHODS AND RESULTS: This report is the first application of the reverse dot-blot (RDB) assay for diagnosis of the nine most common point mutations in the CYP21 gene associated with CAH (P30L, g.659A>G or g.659C>G, I172N, I236N-V237E-M239K, V281L, g.1767-1768insT, Q318X, R356W, P453S). Normal and mutant oligonucleotides spanning these nine mutation sites were spotted onto a nylon membrane. DNA was extracted from dried blood spots, and exons encompassing mutations from samples to be tested were amplified and labeled with biotin-dUTP by PCR. These exons then were hybridized to membrane strips. Signal detection was achieved by chemiluminescence. Thirty clinically confirmed cases that were identified by the Texas Newborn Screening Program were tested. All mutations were subsequently confirmed by automated DNA sequencing. CONCLUSION: The RDB method has the advantages of being accurate and cost-effective for the molecular diagnosis of CYP21 point mutations in CAH. It permits simultaneous detection of a panel of point mutations with only one hybridization per sample and could be automated to study many samples.

X-radiation-induced chromosome breakage in retinoblastoma lymphocytes.

We have examined the spontaneous and X-radiation-induced chromosomal damage in normal humans and in patients with retinoblastoma using the BudR-Giemsa technique in lymphocytes cultured for 48 h. 9 sporadic unilateral non-hereditary cases, 11 hereditary cases (8 bilateral sporadic and 3 unilateral hereditary cases) and 20 healthy individuals were studied simultaneously. No difference in the spontaneous frequency of chromatid and chromosome aberrations was observed between patients and controls. After treatment with 150 rad the frequency of chromosome exchange aberrations was higher in unilateral hereditary cases than the controls (42.0% +/- 5.3 and 22.3% +/- 2.6 respectively; p = 0.05). In bilateral sporadic retinoblastoma 2 different groups were observed. A hypersensitive group showed a significant increment in radiation-induced chromosomal exchange aberrations over the control group (46.2% +/- 5.4 and 24.2% +/- 2.1 respectively; p = 0.01). The other group had a chromosomal exchange frequency similar to normal individuals (26.5% +/- 2.0 and 24.2% +/- 0.4 respectively; p = 0.10). Sporadic unilateral non-hereditary retinoblastoma had an exchange chromosomal aberration frequency similar to control individuals (26.1% +/- 2.8 and 24.6% +/- 2.7 respectively; p greater than 0.10). These results suggest that: There is no relationship between spontaneous chromosome fragility and retinoblastoma. Sporadic unilateral non-hereditary retinoblastoma has normal chromosome sensitivity to X-irradiation. Some hereditary cases of retinoblastoma are sensitive to X-rays while others behave like normals. A mutation or a submicroscopic deletion at a DNA repair locus which is independent of the retinoblastoma gene may cause this radiosensitivity.

Chromosomal radiosensitivity of pig leucocytes in relation to sampling time.

Pig blood cultures were used to analyse the sensitivity to X-rays (measured as frequency of induced dicentrics) of lymphocytes sampled at variable times. By using the BrdU-Giemsa method it was possible to identify the lymphocytes that were performing their first division at early (less than 30% of cells in second division), intermediate (30-50% of cells in second or subsequent divisions) and late stages (more than 50% of cells in second or subsequent divisions). No difference was found in the radiosensitivity of these 3 varieties of lymphocyte. It was also observed that: (a) the combination of radiation followed by BrdU treatment did not increase the clastogenic action of X-rays, (b) X-rays in the dose used in our cultures did not increase the frequency of SCEs, and (c) minor changes in culture conditions probably influence the basal frequency of SCEs.