Modulation of ochratoxin A induced DNA-damage in urothelial cell cultures

Despite good evidence for a genotoxic potential of ochratoxin A (OTA), the mechanism of OTA-induced genotoxicity (direct or indirect?) is still unclear. This calls for a further characterization of OTA-related DNA damage, and investigations of factors that may modulate dose-effect relationships in cells. Since bladder epithelium is a target tissue for the toxicity of OTA, its effects were studied in cultures of human bladder carcinoma (H5637) cells. Cytotoxicity of OTA, assessed by Neutral red (NR) uptake or Alamar-Blue assay, is concentration- and time-dependent: Upon 24 h treatment of 5637 cells, NR uptake is reduced by 50% with OTA concentrations of ≥0.2 microM, but not with 3 h treatment of the cells. Since cytotoxicity of OTA was not affected by addition of xenobiotic metabolizing enzymes (S-9 mix), it appears to be unrelated to biotransformation of the mycotoxin. Also, addition of S-9 mix did not significantly affect the genotoxicity of OTA as studied by alkaline single cell gel electrophoresis (Comet assay). DNA damage was detectable after 3 h treatment of cells at OTA concentrations between 0.1 and 1 microM, and increased further at higher concentrations. The magnitude of OTA-induced DNA damage did not increase with longer treatment times (18, 24 h), probably due to repair processes in the cells. Repair of OTA-induced lesions is quite efficient in kidney (Arch Toxicol 2002, 75, 734–741) and in porcine bladder cells (Föllmann and Lebrun, 2005, Mycotoxin Research, this volume). Interestingly, the genotoxicity of OTA is modulated by the pH of the culture medium, with higher damage at pH 5 compared to pH 7.5. In line with this, uptake studies with tritiated OTA show a higher cellular accumulation of the mycotoxin at pH 5 than in buffer of pH 7.5. Thus, bladder cells exposed to OTA in slightly acidic urine (which facilitates reabsorption) may be at higher risk.     

Effects of ochratoxin A on DNA evaluated<Emphasis Type="Italic">in vitro</Emphasis> with the single cell gel electrophoresis (Comet assay)

In cell cultures of Madin Darby canine kidney (MDCK) cells, the mycotoxin ochratoxin A (OTA) induced single strand breaks (ssb) in a concentration dependent manner detected with the single cell gel electrophoresis (Comet assay). When an external metabolizing enzyme system (S9-mix from rat liver) was added, this genotoxic effect was significantly stronger. By addition of methotrexate (MT), a substrate of the hepatic organic anion transporter, the effect of OTA can be completely blocked at concentrations >100 μM methotrexate.When DNA repair was inhibited by addition of cytosine arabinose (araC) and hydroxyurea (HU), the tail length in the Comet assay increased dramatically and all treated cells showed ssb. A further culture of the damaged cells in the absence of any supplement resulted in a complete repair of the damaged DNA within three hours.Compared with MDCK cells, primary cultured porcine urinary bladder epithelial cells (PUBECs) showed weaker effects in the Comet assay if treated with OTA. The presence of S9-mix did not significantly enhance the response. Methotrexate only partially reduced the OTA-induced effects, because in PUBECs methotrexate induced ssb at high concentrations. If DNA repair was inhibited, also in PUBECs clearly more ssb were induced by OTA, an effect which was reversible.These results demonstrate that OTA induces single strand breaks in vitro. The damaged DNA can be repaired more effectively in primary cultured epithelial cells (PUBECs) compared to cells of a cell line (MDCK cells). By competitive inhibition of OTA uptake, DNA damage can be prevented with suitable substrates.     

Water pH and metabolic parameters in silver catfish (Rhamdia quelen)

This study evaluated the effects of an acute change in water pH (from pH 7.5 to 4.0, 5.0, 6.0, 7.5, 8.0 or 9.0) on several biochemical parameters in juveniles of the silver catfish, Rhamdia quelen. Ammonia levels decreased in the liver and increased in the muscle with increasing water pH. In the kidney, lower ammonia levels were observed at neutral pH. An increase in water pH decreased the glucose, glycogen and lactate levels in the liver and kidney (except for glycogen levels in the kidney and lactate levels in the liver, which presented lower levels at neutral pH). In muscle, the glucose and glycogen levels decreased with increasing water pH, whereas lactate levels tended to be lower at neutral pH. Gill and kidney Na⁺/K⁺-ATPase activities tended to increase in alkaline water, and the highest value was observed in fish exposed to pH 9.0. The optimal levels of the analyzed biochemical parameters occurred at neutral pH. In conclusion, exposure to acidic and alkaline pH changes the metabolic parameters of silver catfish as well as gill Na⁺/K⁺-ATPase activity.     

Validation and application of Drosophila melanogaster as an in vivo model for the detection of double strand breaks by neutral Comet assay

Comet assay under neutral conditions allows detection of DNA double-strand breaks (DSBs), which has consequence to genome instability and carcinogenesis. The present study aims to validate the neutral Comet assay for genotoxicity assessment in Drosophila melanogaster (Oregon R(+)) with three well known DSBs inducers i.e. cyclophosphamide (CP), bleomycin (BLM), cisplatin (CPT) and subsequently its efficacy in detecting DSBs in the organism exposed to a well known environmental chemical, chromium [Cr(VI)]. Third instar larvae of D. melanogaster were fed different concentrations of BLM, CPT and CP (50.0-200.0μg/ml) or Cr(VI) (5.0-20.0μg/ml) mixed standard Drosophila food for 48h. Neutral Comet assay was performed in cells of mid gut and brain from control and treated larvae. Our results show a dose-dependent increase in the migration of DNA in cells of the exposed organisms. A comparison among DNA lesions per mole number of the test chemical in the exposed groups showed that both BLM and CPT induce more DSBs than CP. Interestingly, Cr(VI) at 20.0μg/ml was found to induce significantly increased (p<0.001) DSBs in the exposed organism as compared to the control. The study while validating neutral Comet assay in D. melanogaster suggests its use for in vivo assessment of environmental chemical induced DSBs.     

Metal Interactions with Microbial Biofilms in Acidic and Neutral pH Environments

Microbial biofilms were grown on strips of epoxy-impregnated filter paper submerged at four sites in water contaminated with metals from mine wastes. At two sample stations, the water was acidic (pH 3.1); the other sites were in a lake restored to a near neutral pH level by application of a crushed limestone slurry. During a 17-week study period, planktonic bacterial counts increased from 10¹ to 10³ CFU/ml at all sites. Biofilm counts increased rapidly over the first 5 weeks and then leveled to 10⁴ CFU/cm² in the neutral pH system and 10³ CFU/cm² at the acidic sites. In each case, the biofilms bound Mn, Fe, Ni, and Cu in excess of the amounts adsorbed by control strips covered with nylon filters (pore size, 0.22 μm) to exclude microbial growth; Co bound under neutral conditions but not under acidic conditions. Conditional adsorption capacity constants, obtained graphically from the data, showed that biofilm metal uptake at a neutral pH level was enhanced by up to 12 orders of magnitude over acidic conditions. Similarly, adsorption strength values were usually higher at elevated pH levels. In thin sections of the biofilms, encapsulated bacterial cells were commonly found enmeshed together in microcolonies. The extracellular polymers often contained iron oxide precipitates which generated weak electron diffraction patterns with characteristic reflections for ferrihydrite (Fe₂O₃ · H₂O) at d equaling 0.15 and 0.25 nm. At neutral pH levels, these deposits incorporated trace amounts of Si and exhibited a granular morphology, whereas acicular crystalloids containing S developed under acidic conditions.     

Induction of micronuclei by ochratoxin A is a sensitive parameter of its genotoxicity in cultured cells

In order to better characterize the ochratoxin A (OTA)-induced DNA damage and to further investigate factors which may modulate dose-effect relationships in cells, the induction of micronuclei was studied in V79 Chinese hamster fibroblast cells and in primary cultures of porcine urothelial bladder epithelial cells (PUBEC). OTA was able to induce micronuclei in PUBEC and V79 cells at concentrations below those which were overtly cytotoxic. OTA concentrations between 0.03 and 1 μM caused a dose-dependent increase of micronuclei in V79 cells (up to 3-fold compared to controls); but the lowest tested concentration of 0.01 μM OTA did not induce a higher frequency of micronuclei than in the solvent control, indicative of an apparent threshold. Clear evidence for genotoxic effects was also found in PUBEC cultures treated with OTA concentrations of 1 μM and more, although the dose-effect relationship in PUBEC was more variable for several freshly isolated cell batches, pointing to differences in susceptibility to OTA between bladder cells from different donor animals. The chromosomal genotoxicity of OTA demonstrated in this study is in general accord with previous findings on the induction of clastogenic effects and oxidative DNA damage by OTA. In both cases, the shape of the dose-response curve at very low OTA concentrations supports the existence of a threshold for its genotoxicity. Presented at the 28^th Mykotoxin-Workshop, Bydgoszcz, Poland, May 29–31, 2006     

Regulation of vascular endothelial growth factor binding and activity by extracellular pH

Angiogenesis, the growth of new blood vessels, is regulated by a number of factors, including hypoxia and vascular endothelial growth factor (VEGF). Although the effects of hypoxia have been studied intensely, less attention has been given to other extracellular parameters such as pH. Thus, the present study investigates the consequences of acidic pH on VEGF binding and activity in endothelial cell cultures. We found that the binding of VEGF165 and VEGF121 to endothelial cells increased as the extracellular pH was decreased from 7.5 to 5.5. Binding of VEGF165 and VEGF121 to endothelial extracellular matrix was also increased at acidic pH. These effects were, in part, a reflection of increased heparin binding, because VEGF165 and VEGF121 showed increased retention on heparin-Sepharose at pH 5.5 compared with pH 7.5. Consistent with these findings, soluble heparin competed for VEGF binding to endothelial cells under acidic conditions. However, at neutral pH (7.5) low concentrations of heparin (0.1-1.0 microg/ml) potentiated VEGF binding. Extracellular pH also regulated VEGF activation of the extracellular signal-regulated kinases 1 and 2 (Erk1/2). VEGF165 and VEGF121 activation of Erk1/2 at pH 7.5 peaked after 5 min, whereas at pH 6.5 the peak was shifted to 10 min. At pH 5.5, neither VEGF isoform was able to activate Erk1/2, suggesting that the increased VEGF bound to the cells at low pH was sequestered in a stored state. Therefore, extracellular pH might play an important role in regulating VEGF interactions with cells and the extracellular matrix, which can modulate VEGF activity.     

K+ Uptake by Fermenting Escherichia coli Cells: pH Dependent Mode of the TrkA System Operating

Escherichia coli accumulates K⁺ by means of multiple transportsystems, of which TrkA is the most prominent at neutral and alkalinepH while Kup is major at acidic pH. In the present study, K⁺ uptakewas observed with cells grown under fermentative conditions at an initialpH of 9.0 and 7.3 (the medium pH decreased to 8.4 and 6.8, respectively,during the mid-logarithmic growth phase), washed with distilled water andresuspended in a K⁺ containing medium at pH 7.5 in the presence ofglucose. The kinetics for this K⁺ uptake and the amount of K⁺accumulated by the wild type and mutants having a functional TrkA orKup could confirm that K⁺ uptake by E. coli grown either at pH 9.0or pH 7.3 occurs mainly through TrkA. The following results distinguishpH dependent mode of TrkA operating: (1) K⁺ uptake was inhibited byDCCD in cells grown either at pH 9.0 or pH 7.3, although the stoichiometryof K⁺ influx to DCCD-inhibited H⁺ efflux for bacteria grownat pH 9.0 varied with external K⁺ concentration, but remained constantfor cells grown at pH 7.3; (2) K⁺ uptake was observed with an atpDmutant grown at pH 9.0 but not at pH 7.3; (3) The DCCD-inhibited H⁺efflux was increased 8-fold less by 5 mM K⁺ added into a K⁺ freemedium for bacteria grown at pH 9.0 than that for cells grown at pH 7.3;(4) the DCCD-inhibited ATPase activity of membrane vesicles from bacteriagrown at pH 9.0 was reduced a little in the presence of 100 mM K⁺,but stimulated more than 2.4-fold at pH 7.3.     

Effect of culture pH on the extracellular production of 5-aminolevulinic acid by Rhodobacter sphaeroides from volatile fatty acids

Summary 5-Aminolevulinic acid(ALA) production by Rhodobacter sphaeroides was investigated at various pH with levulinic acid addition using a volatile fatty acids medium prepared from the mandarin orange peel supplemented with glycine. At neutral pH (6.8 and 7.0), extracellular ALA production was up to 16 mM, while low production of ALA(less than 3.5 mM) was observed at acidic pH (lower than 6.5) and less than 3.9 mM of ALA produced at alkaline pH (higher than 7.5). The higher ALA synthase activity observed at neutral pH might enhance the ALA production compared with that observed in acidic and alkaliphilic cultures.     

Ribonuclease activities in rat sympathetic ganglia: Evidence for the presence of an endogenous inhibitor of alkaline ribonuclease

Using³H-labeled rat brain mature RNA as substrate, substantial ribonuclease activity was detected in homogenates of rat superior cervical ganglia with acidic (pH 5.5) and neutral (pH 7.0-7.5) optima. Very little activity could be measured at greater than pH 8. The acidic and neutral activities differed in the optimal conditions required for assay, and showed differential sensitivity to the sulfhydryl blocking agent, N-ethylmaleimide. Only the neutral activity was stimulated, optimally by 2 mM N-ethylmaleimide, and the magnitude of stimulation indicated that the contributing ribonucleases exist largely in a latent form in the ganglion. Ribonucleases in other tissues with neutral pH dependence, known usually as alkaline ribonucleases, are subject to an N-ethylmaleimide-sensitive endogenous inhibitor protein. The existence of a similar inhibitor in rat superior cervical ganglia was indicated by the latency of neutral ribonuclease activity and confirmed by observing the effect of a soluble fraction from the ganglia on the activity of pancreatic ribonuclease A.     

Role of endocytosis and low pH in murine hepatitis virus strain A59 cell entry

Infection by the coronavirus mouse hepatitis virus strain A59 (MHV-A59) requires the release of the viral genome by fusion with the respective target membrane of the host cell. Fusion is mediated by the viral S protein. Here, the entry pathway of MHV-A59 into murine fibroblast cells was studied by independent approaches. Infection of cells assessed by plaque reduction assay was strongly inhibited by lysosomotropic compounds and substances that interfere with clathrin-dependent endocytosis, suggesting that MHV-A59 is taken up via endocytosis and delivered to acidic endosomal compartments. Infection was only slightly reduced in the presence of substances inhibiting proteases of endosomal compartments, precluding that the endocytic uptake is required to activate the fusion potential of the S protein by its cleavage. Fluorescence confocal microscopy of labeled MHV-A59 confirmed that virus is taken up via endocytosis. Bright labeling of intracellular compartments suggests their fusion with the viral envelope. No fusion with the plasma membrane was observed at neutral pH conditions. However, when virus was bound to cells and the pH was lowered to 5.0, we observed a strong labeling of the plasma membrane. Electron microscopy revealed low pH triggered conformational alterations of the S ectodomain. Very likely, these alterations are irreversible because low-pH treatment of viruses in the absence of target membranes caused an irreversible loss of the fusion activity. The results imply that endocytosis plays a major role in MHV-A59 infection and the acidic pH of the endosomal compartment triggers a conformational change of the S protein mediating fusion.     

Uptake of neutral and acidic amino acids by Lemna gibba correlated with the H+-electrochemical gradient at the plasmalemma

Uptake rates of L-alanine, L-serine and L-aspartate and trans-membrane electrical potentials (Δψ) were determined for a pH range in the external medium between 3.5 and 9.0. The proton electrochemical gradients (     ) were calculated from Δψ, pH of the medium, and an assumed cytoplasmic pH of 7.5. At external amino-acid concentrations of 0.1 mol m⁻³, where carrier-mediated uptake dominates total uptake, a linear correlation between uptake rates and     is obtained, which extrapolates to zero uptake at zero     . This corroborates the contention that neutral and acidic amino acids are taken up by Lemna gibba L. by H⁺-cotransport.     

Low-pH-Dependent Fusion of Sindbis Virus with Receptor-Free Cholesterol- and Sphingolipid-Containing Liposomes

There is controversy as to whether the cell entry mechanism of Sindbis virus (SIN) involves direct fusion of the viral envelope with the plasma membrane at neutral pH or uptake by receptor-mediated endocytosis and subsequent low-pH-induced fusion from within acidic endosomes. Here, we studied the membrane fusion activity of SIN in a liposomal model system. Fusion was followed fluorometrically by monitoring the dilution of pyrene-labeled lipids from biosynthetically labeled virus into unlabeled liposomes or from labeled liposomes into unlabeled virus. Fusion was also assessed on the basis of degradation of the viral core protein by trypsin encapsulated in the liposomes. SIN fused efficiently with receptor-free liposomes, consisting of phospholipids and cholesterol, indicating that receptor interaction is not a mechanistic requirement for fusion of the virus. Fusion was optimal at pH 5.0, with a threshold at pH 6.0, and undetectable at neutral pH, supporting a cell entry mechanism of SIN involving fusion from within acidic endosomes. Under optimal conditions, 60 to 85% of the virus fused, depending on the assay used, corresponding to all of the virus bound to the liposomes as assessed in a direct binding assay. Preincubation of the virus alone at pH 5.0 resulted in a rapid loss of fusion capacity. Fusion of SIN required the presence of both cholesterol and sphingolipid in the target liposomes, cholesterol being primarily involved in low-pH-induced virus-liposome binding and the sphingolipid catalyzing the fusion process itself. Under low-pH conditions, the E2/E1 heterodimeric envelope glycoprotein of the virus dissociated, with formation of a trypsin-resistant E1 homotrimer, which kinetically preceded the fusion reaction, thus suggesting that the E1 trimer represents the fusion-active conformation of the viral spike.     

Binding of oligonucleotides to cell membranes at acidic pH.

Antisense oligodeoxynucleotides [oligo(dN)] have the ability to enter living cells and block the expression of specific genes. However, little is known about the mechanism of cellular uptake of oligo(dN). We have found that oligo(dN) can bind to the cell membranes of eukaryotic cells with much greater efficiency under acidic conditions (pH 4.0-4.5) than at neutral pH. The binding appears to be specific to poly nucleic acids since various sizes of oligo(dN), DNA and RNA, but not mononucleotides, compete for the binding. We have identified a 34 kDa membrane protein from T-cells, which binds to oligo(dT) cellulose at pH 4.5 and can be eluted at pH 7.5. This protein fraction blocked the binding of oligo(dN) to living T-cells in a competitive fashion. Our results suggest that eukaryotic cells have a receptor for oligo(dN) at acidic pH and that the 34 kDa dalton protein on the cell membrane may mediate such binding.     

Repair of ochratoxin A-induced DNA damage and modulation of OTA-related genotoxicity by co-incubation with bile acids and methotrexatein vitro

The mycotoxin ochratoxin A (OTA) induced DNA strand breaks in porcine urinary bladder epithelial cells (PUBEC) and in Madin Darby canine kidney (MDCK) cells. A co-incubation with bile acids or methotrexate reduced or even prevented this adverse effect of OTAin vitro. The protective effect is possibly attributable to a decreased OTA uptake in cells, since bile acids and methotrexate are known to share common transport systems such as organic anion transporters (OAT) and/or organic anion transporting polypeptides (OATP) with the mycotoxin. OTA uptake in cells and its modulation can be one factor which determines the extent of adverse effects in different cell types. Another aspect of interest in this regard relates to repair of DNA damage: PUBEC cells are sensitive to OTA-induced damage which is more pronounced when DNA repair is blocked (by cytosine β-D-arabino-furanosid/hydroxyurea). On the other hand, when cells are kept in fresh (toxin-free) medium for 3 h, OTA-induced DNA damage decreased to control levels. Presented at the 26^th Mykotoxin-Workshop in Herrsching, Germany, May 17–19, 2004     

Combinatory effects of citrinin and ochratoxin A in immortalized human proximal tubule cells

Ochratoxin A (OTA) and citrinin (CIT) are two mycotoxins often occurring together in grains and cereals. Although both are nephrotoxic and can induce apoptosis, combination effects have not been examined up to now. Therefore, the aim of this study was to take a close look at the interactions of citrinin and OTA in cultured human proximal tubule-derived cells (IHKE cells). The cytotoxicity of both mycotoxins was studied, measuring the metabolic activity and the cell number. Furthermore, caspase 3-activation as a marker for apoptosis was examined for both mycotoxin alone and in combination. The results show that citrinin had an antagonistic effect on ochratoxin A induced caspase 3-activation in concentrations of 2.5 and 5 μmol/l. Higher concentrations (7.5 and 15 μmol/l) lead to additive effects, lower citrinin concentrations (0.25 and 1 μmol/l) did not show any effect at all. The observed decrease in caspase 3-activity was specific for the combination with OTA, since the combination of citrinin with cisplatin did not show any effect. Citrinin did not influence of the OTA-induced apoptosis when added two hours after applying ochratoxin A. Also the combination of both toxins decreased the uptake of OTA into the cells which might be an explanation for the antagonistic effect of citrinin in certain concentrations. However, the transport into cells can not be the only explanation. so further examinations are necessary. Presented at the 27^th Mykotoxin-Workshop. Dortmund, Germany, June 13–15, 2005.     

Comparative analysis of micronuclei and DNA damage induced by Ochratoxin A in two mammalian cell lines

The fungal toxin, Ochratoxin A (OTA), is a common contaminant in human food and animal feed. The present study evaluated micronucleus (MN) induction by OTA in comparison with its ability to induce cytotoxicity and DNA damage in two mammalian cell lines, CHO-K1-BH(4) Chinese hamster ovary cells and TK6 human lymphoblastoid cells. Micronuclei were evaluated by flow cytometry, cytotoxicity was estimated by relative population doubling (RPD), while direct DNA damage and oxidative DNA damage were measured with the Comet assay, performed without and with digestion by formamidopyrimidine-DNA glycosylase (fpg). For the MN and cytotoxicity measurements, the cell lines were treated for 24h (CHO cells) or 27h (TK6 cells) with 5-25μM OTA in the absence of exogenous metabolic activation. The OTA treatments resulted in concentration-responsive increases in cytotoxicity, with higher concentrations of the agent being more cytotoxic in CHO cells than TK6 cells. 15μM OTA produced positive responses for MN induction and hypodiploid events (a measure of aneugenicity) in both cell lines; this concentration of OTA also produced cytotoxicity near to the recommended limit for the assay (45±5% RPD). A time course assay with TK6 cells indicated that at least 4h of OTA treatment were required to produce a positive MN response. For the Comet assay DNA damage assessments, the cell lines were treated with 5-50μM OTA for 4h. Direct DNA damage was detected in TK6 cells, but not CHO cells, while concentration-related increases in fpg-sensitive sites were detected for both cell lines. The consistent association of oxidative DNA damage with OTA exposure suggests its involvement in producing OTA-induced clastogenicity and aneugenicity; however, based on its detection in TK6 cells direct DNA damage could be involved in any human risk posed by OTA exposure.     

Survival of a Salmonella typhimurium poultry isolate in the presence of propionic acid under aerobic and anaerobic conditions

Propionic acid is commonly found as a fermentation product in the gastrointestinal tracts of food animals and has also been used to limit the microbial contaminants in animal feeds. Because propionic acid is known to have antibacterial activity, the propionic acid encountered by foodborne pathogens during their life cycles may play an important role in inhibiting the survival of the pathogens. The survival patterns of Salmonella typhimurium poultry isolate were determined both in aerobic and anaerobic tryptic soy broth (TSB; pH 5.0 or 7.0) containing various concentrations of propionic acid (0-200 mM). The levels of recovered cells were consistently greater at pH 7.0 compared to those at pH 5.0. For the first 4 days, the levels were significantly decreased by incubation under anaerobic conditions as compared to aerobic condition at pH 7.0 (P<0.05). However, there were fluctuations of cell populations with different patterns depending on both concentrations and growth conditions. To characterize the nature of the capability which allowed the cell multiplication following decreases in cell population during incubation at pH 7.0, the cells isolated from the outgrowth cultures were tested for survival in aerobic or anaerobic TSB (pH 5.0 or pH 7.0) containing propionic acid (50 mM). The outgrowth isolates did not show significant differences in the level of recovered cells in the presence of propionic acid when compared to the wild type strain (P>0.05), suggesting that the cells in the outgrowth cultures did not harbour mutation(s) conferring increased resistance to propionic acid. In addition, the level of recovered cells of isogenic rpoS mutant strain of S. typhimurium was not significantly different from that of the wild type strain in the same assay conditions (P<0.05). The results of this study show that the bactericidal activity of propionic acid on S. typhimurium can be affected by environmental conditions such as acidic pH levels and anaerobiosis in food materials and gastrointestinal tracts. However, S. typhimurium is also able to multiply in the presence of sublethal concentrations of propionic acid at neutral pH during prolonged incubation under both aerobic and anaerobic conditions.     

Dominance of yeast in activated sludge under acidic pH and high organic loading

Flow cytometry-fluorescent in situ hybridization (FC-FISH) was used to investigate the effect of controlled pH and/or varied organic loading on the content of yeast and bacterial cells in an activated sludge system (AS) individually operating in continuous and batch mode for treatment of high-strength industrial wastewater. Specifically, we attempted to develop a yeast-predominant activated sludge system (Y-AS). For the batch-mode AS, bacteria-dominated AS (B-AS) obtained at pH 6.5–7.5 induced higher chemical oxygen demand (COD) removal than Y-AS obtained at acidic pH (5.0–6.0 and 4.0–5.0). For the continuous-mode AS operating at COD loadings of 2.5–2.8 kg COD m⁻³ d⁻¹, it was difficult to achieve a Y-AS solely by controlling the pH level at 7.0 to 5.1 then to 4.1 because bacteria stably accounted for greater than 98% of the total cells, regardless of the pH levels. Therefore, the effects of varied COD loadings (2.1, 8.7 and 21.0 kg COD m⁻³ d⁻¹) on continuous-mode AS operation at acidic pH (4.5) was investigated. Both acidic pH and high COD loading levels were found to be prerequisites for yeast to dominate the sludge microbial community in the continuous-mode AS.     

Acid-stable cytochromes in ferrous ion oxidizing cell-free preparations from Thiobacillus ferrooxidans

Abstract Cell-free preparations from ferrous ion- and sulfur-grown Thiobacillus ferrooxidans prepared under neutral (pH 7.5) or acidic conditions (pH 2.0) were compared. Under neutral conditions the ferrous ion-oxidizing system of T. ferrooxidans was membrane-bound. At acidic conditions, the enzyme system became partially solubilized. In ferrous ion-oxidizing membranes of ferrous ion-grown cells (neutral conditions) a₁-, c- and b-type cytochromes were present. The acidic preparations contained only cytochrome a₁ and c. At least three acid-stable c-type cytochromes (M_r 60 000, 30 000 and 25 000), an acid-stable protein with non-convalently bound heme group (M_r probably rusticyanin, were detected in ferrous ion oxidizing preparations. Membranes of sulfur-grown cells prepared under neutral conditions still oxidized ferrous ions, and contained a₁-, b-, c- and in addition an aa₃-type cytochrome. Cytochrome b and aa₃ were acid-labile.