Using fluorescent promoter-reporters to study sugar utilization control in Bifidobacterium longum NCC 2705.

Bifidobacteria are amongst the first bacteria to colonize the human gastro-intestinal system and have been proposed to play a crucial role in the development of the infant gut since their absence is correlated to the development of diseases later in life. Bifidobacteria have the capacity to metabolize a diverse range of (complex) carbohydrates, reflecting their adaptation to the lower gastro-intestinal tract. Detailed understanding of carbohydrate metabolism regulation in this genus is of prime importance and availability of additional genetic tools easing such studies would be beneficial. To develop a fluorescent protein-based reporter system that can be used in B. longum NCC 2705, we first selected the most promising fluorescent protein out of the seven we tested (i.e., mCherry). This reporter protein was then used to study the carbohydrate mediated activation of PBl1518 and PBl1694, two promoters respectively predicted to be controlled by the transcriptional factors AraQ and AraU, previously suggested to regulate arabinose utilization and proposed to also act as global transcriptional regulators in bifidobacteria. We confirmed that in B. longum NCC 2705 the AraQ controlled promoter (PBl1518) is induced strongly by arabinose and established that the AraU controlled promoter (PBl1694) was mostly induced by the hexoses galactose and fructose. Combining the mCherry reporter system with flow cytometry, we established that NCC 2705 is able to co-metabolize arabinose and glucose while galactose was only consumed after glucose exhaustion, thus illustrating the complexity of different carbohydrate consumption patterns and their specific regulation in this strain.

Optical analysis of glutamate spread in the neuropil.

Fast synaptic communication uses diffusible transmitters whose spread is limited by uptake mechanisms. However, on the submicron-scale, the distance between two synapses, the extent of glutamate spread has so far remained difficult to measure. Here, we show that quantal glutamate release from individual hippocampal synapses activates extracellular iGluSnFr molecules at a distance of >1.5 µm. 2P-glutamate uncaging near spines further showed that alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-Rs and N-methyl-D-aspartate (NMDA)-Rs respond to distant uncaging spots at approximately 800 and 2000 nm, respectively, when releasing the amount of glutamate contained in approximately five synaptic vesicles. The uncaging-induced remote activation of AMPA-Rs was facilitated by blocking glutamate transporters but only modestly decreased by elevating the recording temperature. When mimicking release from neighboring synapses by three simultaneous uncaging spots in the microenvironment of a spine, AMPA-R-mediated responses increased supra-additively. Interfering with extracellular glutamate diffusion through a glutamate scavenger system weakly reduced field synaptic responses but not the quantal amplitude. Together, our data suggest that the neuropil is more permissive to short-range spread of transmitter than suggested by theory, that multivesicular release could regularly coactivate nearest neighbor synapses and that on this scale glutamate buffering by transporters primarily limits the spread of transmitter and allows for cooperative glutamate signaling in extracellular microdomains.

Carbohydrate-controlled serine protease inhibitor (serpin) production in Bifidobacterium longum subsp. longum.

The Serine Protease Inhibitor (serpin) protein has been suggested to play a key role in the interaction of bifidobacteria with the host. By inhibiting intestinal serine proteases, it might allow bifidobacteria to reside in specific gut niches. In inflammatory diseases where serine proteases contribute to the innate defense mechanism of the host, serpin may dampen the damaging effects of inflammation. In view of the beneficial roles of this protein, it is important to understand how its production is regulated. Here we demonstrate that Bifidobacterium longum NCC 2705 serpin production is tightly regulated by carbohydrates. Galactose and fructose increase the production of this protein while glucose prevents it, suggesting the involvement of catabolite repression. We identified that di- and oligosaccharides containing galactose (GOS) and fructose (FOS) moieties, including the human milk oligosaccharide Lacto-N-tetraose (LNT), are able to activate serpin production. Moreover, we show that the carbohydrate mediated regulation is conserved within B. longum subsp. longum strains but not in other bifidobacterial taxons harboring the serpin coding gene, highlighting that the serpin regulation circuits are not only species- but also subspecies- specific. Our work demonstrates that environmental conditions can modulate expression of an important effector molecule of B. longum, having potential important implications for probiotic manufacturing and supporting the postulated role of serpin in the ability of bifidobacteria to colonize the intestinal tract.

The circadian dynamics of the hippocampal transcriptome and proteome is altered in experimental temporal lobe epilepsy.

Gene and protein expressions display circadian oscillations, which can be disrupted in diseases in most body organs. Whether these oscillations occur in the healthy hippocampus and whether they are altered in epilepsy are not known. We identified more than 1200 daily oscillating transcripts in the hippocampus of control mice and 1600 in experimental epilepsy, with only one-fourth oscillating in both conditions. Comparison of gene oscillations in control and epilepsy predicted time-dependent alterations in energy metabolism, which were verified experimentally. Although aerobic glycolysis remained constant from morning to afternoon in controls, it increased in epilepsy. In contrast, oxidative phosphorylation increased in control and decreased in epilepsy. Thus, the control hippocampus shows circadian molecular remapping, which is altered in epilepsy. We suggest that the hippocampus operates in a different functioning mode in epilepsy. These alterations need to be considered when studying epilepsy mechanisms, designing drug treatments, and timing their delivery.

A novel approach to genetic engineering of T-cell subsets by hematopoietic stem cell infection with a bicistronic lentivirus.

Lentiviral modification of hematopoietic stem cells (HSCs) paved the way for in vivo experimentation and therapeutic approaches in patients with genetic disease. A disadvantage of this method is the use of a ubiquitous promoter leads not only to genetic modification of the leukocyte subset of interest e.g. T-cells, but also all other subsequent leukocyte progeny of the parent HSCs. To overcome this limitation we tested a bicistronic lentivirus, enabling subset specific modifications. Designed novel lentiviral constructs harbor a global promoter (mPGK) regulating mCherry for HSCs selection and a T-cell specific promoter upstream of eGFP. Two T-cell specific promoters were assessed: the distal Lck-(dLck) and the CD3δ-promoter. Transduced HSCs were FACS sorted by mCherry expression and transferred into sublethally irradiated C57/BL6 mice. Successful transplantation and T-cell specific expression of eGFP was monitored by peripheral blood assessment. Furthermore, recruitment response of lentiviral engineered leukocytes to the site of inflammation was tested in a peritonitis model without functional impairment. Our constructed lentivirus enables fast generation of subset specific leukocyte transgenesis as shown in T-cells in vivo and opens new opportunities to modify other HSCs derived subsets in the future.

Influence of Ambient Temperature and Light-curing Moment on Polymerization Shrinkage and Strength of Resin Composite Cements.

OBJECTIVE:: The purpose of this study was to establish a clinically appropriate light-curing moment for resin composite cements while achieving the highest indirect tensile strength and lowest polymerization shrinkage. METHODS AND MATERIALS:: Polymerization shrinkage of seven resin composite cements (Multilink Automix, Multilink Speed Cem, RelyX Ultimate, RelyX Unicem 2 Automix, Panavia V5, Panavia SA plus, VITA Adiva F-Cem) was measured at ambient temperatures of 23°C and 37°C. Testing was done for autopolymerized and light-cured specimens after light application at either 1, 5, or 10 minutes after mixing. Indirect tensile strength of all cements was measured after 24 hours of storage at temperatures of 23°C and 37°C, for autopolymerized and light-cured specimens after light application 1, 5, or 10 minutes after mixing. To illustrate filler size and microstructures, SEM images of all cements were captured. Statistical analysis was performed with one-way ANOVA followed by post hoc Fisher LSD test ( α=0.05). RESULTS:: Final polymerization shrinkage of the resin composite cements ranged from 3.2% to 7.0%. An increase in temperature from 23°C to 37°C as well as the light-curing moment resulted in material dependent effects on the polymerization shrinkage and indirect tensile strength of the cements. Polymerization shrinkage of the cements did not correlate with the indirect tensile strength of the cement in the respective groups. Highest indirect tensile strengths were observed for the materials containing a homogeneous distribution of fillers with a size of about 1 µm (Multilink Automix, Panavia V5, VITA Adiva F-Cem). CONCLUSION:: The magnitude of the effect of light-curing moment and temperature increase on polymerization shrinkage and indirect tensile strength of resin composite cements is material dependent and cannot be generalized.

Wound-Healing potential of Sebastiania hispida (Mart) Pax (Euphorbiaceae) ointment compared to low power laser in rats / Potencial cicatrizante da pomada de Sebastiania hispida (Mart) Pax (Euphorbiaceae) comparado ao laser de baixa potência

Abstract Impaired wound healing represents a serious complication in some pathologies and the use of plant extracts has proved to improve tissue repair. The objectives of this study were to evaluate the healing potential of the ointment of Sebastiana hispida compared with Aluminum-Gallium Indium-Phosphide Laser (InGaAlP) in surgically induced wounds in rats and to perform the phytochemical analysis. The phytochemical analysis was performed in the classic way and also by HPLC. A controlled study was developed with 80 rats (200-250 g) in which a linear excision was performed in the dorsal region after shaving, measuring 2 cm × 1 cm (epidermis and dermis) exposing the muscle fascia. The rats were randomly divided into four groups of twenty animals each. The experimental groups (n = 5) were G1 (Saline); G2 (crude methanol plant extract 2% + Carbopol Gel 98%); G3 (crude methanol plant extract 2% + lanolin/vaseline) and G4 (laser). The incision healing processes were monitored during 3, 7, 14 and until 21 days after excision. The histologic parameters evaluated were Collagen fiber types, microscopic examination and neovascularization. There was a significant increase in the deposition of collagen fibers, as evidenced by a better organized epithelial tissue, keratinized and showing greater proliferation of new blood vessels in the inflammatory phase in the group treated with both the extract and laser. The results were correlated to the phenolic derivatives found after qualitative and quantitative analysis. These compounds were considered responsible for the healing process. The topical treatment with S. hispida leaves, in the two different formulations, was more effective than the application of the laser (Ingan ALP) 660 nm in the model used.

Resumo A cicatrização deficiente representa uma complicação grave em algumas patologias e o uso de extratos de plantas tem demonstrado melhora no processo de reparação tecidual. O objetivo deste estudo foi avaliar o potencial cicatrizante da pomada de Sebastiania hispida comparado ao Laser Indio Phosphide-Gallium-Aluminum (InGaAlP) em feridas cutâneas induzidas cirurgicamente em ratos e realizar analise fitoquímica. A análise fitoquímica foi via clássica e por CLAE. O experimento foi desenvolvido com 80 ratos (200-250 g), divididos aleatoriamente em quatro grupos (n = 5): G1 (salina); G2 (extrato bruto metanólico 2% + Carbopol Gel 98%); G3 (extrato bruto metanólico 2% + lanolina/vaselina) e G4 (laser). Após a tricotomia da região dorsal realizou uma excisão linear, medindo 2 cm × 1 cm (epiderme e derme), expondo a fáscia muscular. Em sequência os tratamentos e o monitoramento do processo de cicatrização das feridas cirúrgicas ocorreu nos tempos de: 3, 7, 14 e até 21 dias após a excisão. Os parâmetros histológicos avaliados foram: tipos de fibras colágenas, avaliação morfológica e neovascularização. Houve uma deposição significativa de fibras colágenas, evidenciado por um tecido epitelial mais bem organizado, queratinizado e mostrando uma maior proliferação de novos vasos sanguíneos na fase inflamatória do grupo tratado com o extrato e o laser. A eficiência do processo de cicatrização pode estar relacionada com a presença de compostos fenólicos e derivados detectados na análise qualitativa e quantitativa. A utilização do tratamento tópico com as duas formulações diferentes de S. hispida foram mais eficazes do que a aplicação do Laser (InGaAlP) no modelo utilizado.

Wound-Healing potential of Sebastiania hispida (Mart.) Pax (Euphorbiaceae) ointment compared to low power laser in rats.

Impaired wound healing represents a serious complication in some pathologies and the use of plant extracts has proved to improve tissue repair. The objectives of this study were to evaluate the healing potential of the ointment of Sebastiana hispida compared with Aluminum-Gallium Indium-Phosphide Laser (InGaAlP) in surgically induced wounds in rats and to perform the phytochemical analysis. The phytochemical analysis was performed in the classic way and also by HPLC. A controlled study was developed with 80 rats (200-250 g) in which a linear excision was performed in the dorsal region after shaving, measuring 2 cm × 1 cm (epidermis and dermis) exposing the muscle fascia. The rats were randomly divided into four groups of twenty animals each. The experimental groups (n = 5) were G1 (Saline); G2 (crude methanol plant extract 2% + Carbopol Gel 98%); G3 (crude methanol plant extract 2% + lanolin/vaseline) and G4 (laser). The incision healing processes were monitored during 3, 7, 14 and until 21 days after excision. The histologic parameters evaluated were Collagen fiber types, microscopic examination and neovascularization. There was a significant increase in the deposition of collagen fibers, as evidenced by a better organized epithelial tissue, keratinized and showing greater proliferation of new blood vessels in the inflammatory phase in the group treated with both the extract and laser. The results were correlated to the phenolic derivatives found after qualitative and quantitative analysis. These compounds were considered responsible for the healing process. The topical treatment with S. hispida leaves, in the two different formulations, was more effective than the application of the laser (Ingan ALP) 660 nm in the model used.

Microbial Toluene Removal in Hypoxic Model Constructed Wetlands Occurs Predominantly via the Ring Monooxygenation Pathway.

In the present study, microbial toluene degradation in controlled constructed wetland model systems, planted fixed-bed reactors (PFRs), was queried with DNA-based methods in combination with stable isotope fractionation analysis and characterization of toluene-degrading microbial isolates. Two PFR replicates were operated with toluene as the sole external carbon and electron source for 2 years. The bulk redox conditions in these systems were hypoxic to anoxic. The autochthonous bacterial communities, as analyzed by Illumina sequencing of 16S rRNA gene amplicons, were mainly comprised of the families Xanthomonadaceae, Comamonadaceae, and Burkholderiaceae, plus Rhodospirillaceae in one of the PFR replicates. DNA microarray analyses of the catabolic potentials for aromatic compound degradation suggested the presence of the ring monooxygenation pathway in both systems, as well as the anaerobic toluene pathway in the PFR replicate with a high abundance of Rhodospirillaceae. The presence of catabolic genes encoding the ring monooxygenation pathway was verified by quantitative PCR analysis, utilizing the obtained toluene-degrading isolates as references. Stable isotope fractionation analysis showed low-level of carbon fractionation and only minimal hydrogen fractionation in both PFRs, which matches the fractionation signatures of monooxygenation and dioxygenation. In combination with the results of the DNA-based analyses, this suggests that toluene degradation occurs predominantly via ring monooxygenation in the PFRs.

Modification of the technical properties of Lactobacillus johnsonii NCC 533 by supplementing the growth medium with unsaturated fatty acids.

The aim of this study was to investigate the influence of supplementing growth medium with unsaturated fatty acids on the technical properties of the probiotic strain Lactobacillus johnsonii NCC 533, such as heat and acid tolerance, and inhibition of Salmonella enterica serovar Typhimurium infection. Our results showed that the membrane composition and morphology of L. johnsonii NCC 533 were significantly changed by supplementing a minimal Lactobacillus medium with oleic, linoleic, and linolenic acids. The ratio of saturated to unsaturated plus cyclic fatty acids in the bacterial membrane decreased by almost 2-fold when minimal medium was supplemented with unsaturated fatty acids (10 µg/ml). The subsequent acid and heat tolerance of L. johnsonii decreased by 6- and 20-fold when the strain was grown in the presence of linoleic and linolenic acids, respectively, compared with growth in oleic acid (all at 10 µg/ml). Following acid exposure, significantly higher (P < 0.05) oleic acid content was detected in the membrane when growth medium was supplemented with linoleic or linolenic acid, indicating that saturation of the membrane fatty acids occurred during acid stress. Cell integrity was determined in real time during stressed conditions using a fluorescent viability kit in combination with flow cytometric analysis. Following heat shock (at 62.5°C for 5 min), L. johnsonii was unable to form colonies; however, 60% of the bacteria showed no cell integrity loss, which could indicate that the elevated heat inactivated vital processes within the cell, rendering it incapable of replication. Furthermore, L. johnsonii grown in fatty acid-enriched minimal medium had different adhesion properties and caused a 2-fold decrease in S. enterica serovar Typhimurium UK1-lux invasion of HT-29 epithelial cells compared with bacteria grown in minimal medium alone. This could be related to changes in the hydrophobicity and fluidity of the membrane. Our study shows that technical properties underlying probiotic survivability can be affected by nutrient composition of the growth medium.

Regulatory T cells in peripheral blood, lymph node, and thyroid tissue in patients with medullary thyroid carcinoma.

BACKGROUND: Immunological response of the human body is controlled by the suppressive characteristics of regulatory T cells (Tregs). In various diseases a change in the number of Tregs is evident. For example, whereas Tregs are reduced in auto-immunological processes, an increase of Tregs is found with various malignant tumors. Regarding medullary thyroid carcinoma (MTC) no such studies have been performed to date. METHODS: Expression of CD4 and CD25 in CD45+ leukocytes from blood and lymph nodes was studied by flow cytometry in patients with MTC and patients with benign goiter. We also examined the marker forkhead box P3 (FoxP3), an intracellular transcription factor, which is supposed to be the most specific marker for Tregs. Immunohistochemical staining for FoxP3 was performed on lymph node and thyroid tissue. RESULTS: The number of FoxP3+ lymphocytes in peripheral blood was significantly higher in patients with MTC than in controls (p = 0.02). This result was confirmed immunohistochemically in lymph node and thyroid tissue, as well as in carcinoma tissue. No difference in CD4+CD25+ lymphocytes was observed between the two groups. After clinical staging (International Union against Cancer-UICC-stages) of MTC patients, triplication of FoxP3+ lymphocytes could be observed from MTC < UICC II to MTC > UICC II. CONCLUSIONS: An increase of FoxP3+ lymphocytes could be shown in peripheral blood of patients with MTC but not in patients with benign goiter; this increase also correlates with findings in lymph nodes and thyroid gland. The number of FoxP3+ cells correlated with the patients' prognosis. Therefore, FoxP3+ lymphocytes are a good diagnostic criterion for malignancy in patients with medullary thyroid carcinoma, and their presence at staging may influence therapeutic decisions.

Reconstitution conditions for dried probiotic powders represent a critical step in determining cell viability.

AIMS: Resuscitation of dried cultures represents a critical control point in obtaining active and effective probiotic strains. This study examined the effects of various rehydration conditions on the viability of Bifidobacterium longum NCC3001 and Lactobacillus johnsonii La1. METHODS AND RESULTS: Reconstitution conditions for these strains were optimized using a multivariate experimental design approach. Furthermore, using flow cytometry, the cell integrity was followed during reconstitution. By adjusting the pH, availability of a metabolizable sugar, reconstitution duration, powder matrix and ratio of powder to reconstitution solution, the recovery of Bif. longum NCC3001 and Lact. johnsonii La1 following reconstitution was increased eight- and two-fold, respectively, over standard reconstitution in maximum recovery diluent. It was shown that pH had a significant effect on the recovery of Bif. longum NCC3001 and Lact. johnsonii La1. CONCLUSIONS: The recovery of dried probiotic cultures is greatly dependent on the reconstitution conditions. The maximum recovery of 11.7 (10)log CFU g(-1) Bif. longum NCC3001 was achieved at 30-min reconstitution at pH 8, in the presence of 2% L-arabinose and a ratio of 1:100 of powder to diluent. Lact. johnsonii La1 showed highest recovery (9.3 (10)log CFU g(-1)) after reconstitution, when mixed with maltodextrin at pH 4. SIGNIFICANCE AND IMPACT OF THE STUDY: To achieve accurate viable probiotic numbers from dried probiotic cultures, the reconstitution conditions should be optimized for the strain used.

Reduction of excess sludge production using mechanical disintegration devices.

The usability of mechanical disintegration techniques for the reduction of excess sludge production in the activated sludge process was investigated. Using three different disintegration devices (ultrasonic homogeniser, stirred media mill, high pressure homogeniser) and different operational parameters of the disintegration, the effect of mechanical disintegration on the excess sludge production and on the effluent quality was studied within a continuously operated, laboratory scale wastewater treatment system with pre-denitrification. Depending on the operational conditions and the disintegration device used, a reduction of excess sludge production of up to 70% was achieved. A combination of mechanical disintegration with a membrane bioreactor process with high sludge age is more energy effective concerning reduction of sludge production than with a conventional activated sludge process at lower sludge ages. Depending on the disintegration parameters, the disintegration has no, or only minor, negative effect on the soluble effluent COD and on the COD-removal capacity of the activated sludge process. Nitrogen-removal was slightly deteriorated by the disintegration, whereas the system used was not optimised for nitrogen removal before disintegration was implemented.

Investigation and assessment of sludge pre-treatment processes.

The pre-treatment of sludges by disintegration will result in a number of changes in sludge properties. Floc destruction as well as cell disintegration will occur. This leads to an increase of soluble substances and fine particles. Furthermore, biochemical reactions may appear during or immediately after disintegration. The influence of disintegration of excess sludge on anaerobic digestion was studied in full scale. A stirred ball mill, an ultrasound disintegrator, a lysate centrifuge and ozone treatment were used. The results of the degradation process were compared to a reference system without pre-treatment. An enhancement of the degree of degradation of 7.4% to 20% was observed. The pollution of sludge water as well as the dewatering properties of the digested sludge were investigated. COD and ammonia in the sludge water were increased and a higher polymer demand was observed while the solid content after dewatering stayed almost unchanged. Based on these results the cost effectiveness has been assessed taking into account different conditions (size of WWTP, cost for disposal, etc.). Capital and energy costs are the main factors while the decrease in disposal costs due to the reduced amount of sludge is the main profit factor.

Measurement of glutathione in activated sludges.

Thermal, electric, mechanical or oxidative stress seem a promising way to reduce the production of excess activated sludge during biological wastewater treatment. However, the adaptation and the resistance of the sludge microbial ecosystem to stress conditions is a major question as it may definitively limit the effect of some treatments. Defence mechanisms developed by aerobic organisms, in particular, in response to oxidative stress involve various antioxidant activities and compounds such as glutathione. An HPLC method was developed for measuring reduced and total glutathione (GSH and GSHt) in perchloric acid sludge extracts. The method was sensitive, highly specific and validated for linearity, precision and recovery. Considering the extraction yield and the oxidation of GSH during extract storage, the measured GSH concentration was estimated to represent 60% of the GSH content from activated sludges. GSHt ranged from 0.32 to 3.34micromolg(-1) volatile solids and the GSH/GSHt ratio ranged from 32% to 91%. Measurements performed on sludges stressed in precise conditions selected to reach a reduction of sludge production showed a decrease of GSH and GSHt concentrations with thermal, mechanical, electric and ozone stress.

Anaerobic degradation of organic materials--significance of the substrate surface area.

In anaerobic degradation of substrates containing mainly particulate organic matter, solids hydrolysis is rate-limiting. In these investigations, the particle size of various substrates was reduced by comminution to support hydrolysis. Two positive effects of comminution were observed. For substrates with high fibre content, which are particularly resistant to biodegradation, a significant improvement of the degradation degree was observed as a result of comminution. Secondly, for all substrates tested, and particularly for those rich in fibres, the degradation rate of comminuted samples was significantly higher. The first reason for both effects is an increase of the sample surface area. Several methods for measuring the specific surface area of organic materials, including particle size analysis, Nitrogen-adsorption and enzyme adsorption, were used and compared for the purpose of this study, where the surface area accessible to microbial enzymes is critical. The significance of the surface area in anaerobic degradation of particulate substrates was investigated through a kinetic model where the hydrolysis rate was based on the sample surface area. Good agreements were obtained between model and experiments carried out with samples of various specific surface areas. These results reinforced the significance of the sample area in anaerobic degradation processes. However, other effects of comminution responsible for the increased degradation degree and degradation rate were identified and discussed. These include: the increase of dissolved compounds due to cell rupture, exposition of surface areas previously inacessible for microbial degradation, and alteration of the sample structure such as the lignin-cellulose arrangements.

Initiation of anaerobic degradation of p-cresol by formation of 4-hydroxybenzylsuccinate in desulfobacterium cetonicum.

The anaerobic bacterium Desulfobacterium cetonicum oxidized p-cresol completely to CO(2) with sulfate as the electron acceptor. During growth, 4-hydroxybenzylsuccinate accumulated in the medium. This finding indicated that the methyl group of p-cresol is activated by addition to fumarate, analogous to anaerobic toluene, m-xylene, and m-cresol degradation. In cell extracts, the formation of 4-hydroxybenzylsuccinate from p-cresol and fumarate was detected at an initial rate of 0.57 nmol min(-1) (mg of protein)(-1). This activity was specific for extracts of p-cresol-grown cells. 4-Hydroxybenzylsuccinate was degraded further to 4-hydroxybenzoyl-coenzyme A (CoA), most likely via beta-oxidation. 4-Hydroxybenzoyl-CoA was reductively dehydroxylated to benzoyl-CoA. There was no evidence of degradation of p-cresol via methyl group oxidation by p-cresol-methylhydroxylase in this bacterium.

Prospects and problems of sludge pre-treatment processes.

Pre-treatment processes have been developed in order to improve subsequent sludge treatment and disposal. Disintegration of sludge solids in the aqueous phase changes the sludge structure and solubilizes organic matter. This paper provides an overview of the applications of wet disintegration in wastewater and sludge treatment. Applied disintegration techniques such as mechanical, thermal, chemical and biological methods are briefly described. The methods are compared regarding energy consumption, operational reliability and stage of development for application on wastewater treatment plants. Mechanical and thermal methods appear to be most suitable at this stage. The effects of pre-treatment on subsequent sludge treatment processes and the wastewater treatment are described. The performance of various methods is assessed. For the improvement of stabilization, mechanical and ozone treatment as well as thermal treatment perform best. Dewatering can be enhanced by thermal and freeze/thaw treatment. All methods show positive effects in the reduction of the number of pathogens. Pre-treatment leads to secondary effects like the generation of recalcitrant compounds and odor, which is mainly a problem of thermal and ozone treatment. The evaluation of capital and operational costs is difficult, because of the lack of full-scale experience. Especially thermal, freeze/thaw and biological treatments can be realized at low costs if the conditions are appropriate. Nevertheless, the economic efficiency has to be investigated critically for each individual application.

Initial steps in the fermentation of 3-hydroxybenzoate by Sporotomaculum hydroxybenzoicum.

The anaerobic bacterium Sporotomaculum hydroxybenzoicum ferments 3-hydroxybenzoate to acetate, butyrate, and CO2. 3-Hydroxybenzoate was activated to 3-hydroxybenzoyl-CoA in a CoA-transferase reaction with acetyl-CoA or butyryl-CoA as CoA donors. 3-Hydroxybenzoyl-CoA was reductively dehydroxylated, forming benzoyl-CoA. This reaction was measured in cell-free extracts with cob(I)alamin as low-potential electron donor. No evidence was obtained that cob(I)alamin is the physiological electron donor; however, inhibitor studies indicated involvement of a strong nucleophile in the reaction. Benzoate was degraded by dense cell suspensions without a lag phase until an in situ deltaG' value <-25 kJ mol(-1) was reached. Benzoyl-CoA reductase was not detected. Enzyme activities for all reaction steps from glutaryl-CoA to butyryl-CoA, and ATP formation via acetate kinase were detected in cell-free extracts. Glutaconyl-CoA decarboxylase is likely to act as a primary sodium ion pump.