Potential application of catalase-peroxidase from Comamonas terrigena N3H in the biodegradation of phenolic compounds

Comamonas terrigena N3H is a gram-negative rod-shaped bacterium that was isolated from contaminated soil in Slovakia. This bacterium showed remarkable biodegradation properties. We investigated the expression and functioning of two catalase isozymes in this bacterium. The typical catalase could be induced by cadmium ions, whereas the catalase-peroxidase enzyme was constitutively expressed. Since C. terrigena lacks the key enzyme for complete degradation of phenols (phenolhydroxylase), we analysed the possible removal of phenol by the two catalases of this bacterium. Addition of phenol to the culture medium led to increased expression of the catalase-peroxidase. Applying oxidative stress prior to phenol administration markedly induced the expression of the typical catalase, irrespective of the nature of the added agent. Thus, the rate of phenol degradation is rather reduced under these conditions, while growth of the cells is not impaired. We concluded that phenol peroxidation in C. terrigena can be largely attributed to the action of a catalase-peroxidase. The potential application of this enzyme in the removal of phenol from the environment is discussed.     

The limits of phosphorus removal in wetlands

The phosphorus concentrations exported from wetlands are explored via data and a first order model. The graph of outlet concentration versus areal phosphorus loading is used to display these data and the model. For a given wetland, data and models show that P concentrations show an S curve response to increasing P loadings. The lower plateau is the background concentration and the upper plateau is the inlet concentration. The position of the ascending limb between the two plateaus is positioned differently for different wetlands. Phosphorus (P) removal in wetlands is often typified by a stable decreasing gradient of P concentrations from inlet to outlet, and an accompanying stable decreasing gradient in P assimilation. Limits to removal are inherent in the physical, chemical and biological processes. A lower outlet concentration limit exists because of the P cycle in the un-impacted wetland. The loading at which the outlet concentration departs from background, the lower knee in the loading curve, varies from wetland to wetland. An upper outlet concentration limit is imposed by the source concentration for extremely high inflows. The first order mass balance equation interpolates between these limits. The model gives further insights about performance within an overall envelope. The water carrying capacity of the wetland precludes flows in excess of the hydraulic conveyance capacity, thus limiting the possible P loadings to the system. Conversely, extremely low hydraulic loadings cause the wetland to be dominated by atmospheric additions and losses. The central tendency of inter-system data in the North American Database is shown to be inadequate to draw generalized conclusions about ecosystem processes in an individual wetland. The proposed one gram rule of Richardson, et al. (1997) is shown to be an over-simplification.     

Floral nectaries, nectar production dynamics and chemical composition in six ipomoea species (convolvulaceae) in relation to pollinators

BACKGROUND AND AIMS: Floral nectaries and nectar features were compared between six Argentinian Ipomoea species with differences in their pollinator guilds: I. alba, I. rubriflora, I. cairica, I. hieronymi var. hieronymi, I. indica, and I. purpurea. METHODS: Pollinators were recorded in natural populations. The morpho-anatomical study was carried out through scanning electron and light microscopy. Nectar sugars were identified via gas chromatography. Nectar production and the effect of its removal on total nectar sugar amount were determined by using sets of bagged flowers. KEY RESULTS: Hymenopterans were visitors of most species, while hummingbirds visited I. rubriflora and sphingids I. alba. All the species had a vascularized discoidal nectary surrounding the ovary base with numerous open stomata with a species-specific distribution. All nectar samples contained amino acids and sugars. Most species had sucrose-dominant nectars. Flowers lasted a few hours. Mean nectar sugar concentration throughout the lifetime of the flower ranged from 34.28 to 39.42 %, except for I. cairica (49.25 %) and I. rubriflora (25.18 %). Ipomoea alba had the highest nectar volume secreted per flower (50.12 microL), while in the other taxa it ranged from 2.42 to 12.00 microL. Nectar secretion began as soon as the flowers opened and lasted for a few hours (in I. purpurea, I. rubriflora) or it was continuous during the lifetime of the flower (in the remaining species). There was an increase of total sugar production after removals in I. cairica, I. indica and I. purpurea, whereas in I. alba and I. rubriflora removals had no effect, and in I. hieronymi there was a decrease in total sugar production. CONCLUSIONS: The chemical composition, production dynamics and removal effects of nectar could not be related to the pollinator guild of these species. Flower length was correlated with nectary size and total volume of nectar secreted, suggesting that structural constraints may play a major role in the determination of nectar traits of these species.     

Cold ethanol fractionation and heat inactivation of hepatitis a virus during manufacture of albumin from human plasma

The purpose of the present study was to examine the efficacy and mechanism of fraction IV cold ethanol fractionation and pasteurization (60°C heat treatment for 10h), involved in the manufacture of albumin from human plasma, in the removal and/or inactivation of the hepatitis A virus (HAV). Samples from the relevant stages of the production process were spiked with HAV and the amount of virus in each fraction then quantified using a 50% tissue culture infectious dose (TCID₅₀). HAV was effectively partitioned from albumin during the fraction IV cold ethanol fractionation with a log reduction factor of 3.43. Pasteurization was also found to be a robust and effective step in inactivating HAV, where the titers were reduced from an initial titer of 7.60 log TCID₅₀ to undetectable levels within 5 h of treatment. The log reduction factor achieved during pasteurization was≽4.76. Therefore, the current results indicate that the production process for albumin has sufficient HAV reducing capacity to achieve a high margin of virus safety.     

Behavior of microbial communities developed in the presence/reduced level of soluble microbial products

Soluble microbial products (SMP) are organics produced by microorganisms as they degrade substrates. The available literature does not reveal how SMP affect and regulate microbial activities. In this study, we monitored variations in pH, dissolved oxygen concentration, soluble biological and chemical oxygen demands (sBOD₅ and sCOD) as a measure of microbial activity in synthetic wastewater. Aerobic degradation tests were carried out under the following conditions: aeration, 1,500 cm³ /min; initial sBOD₅, 515±5 mg/l; initial sCOD, 859±6 mg/l; initial biomass concentration (defined as mixed liquor suspended solids), 1,200±25 mg/l; sludge retention time, 24 h; and temperature, 20±1°C. The study involved non-acclimated biomass (R0 flora), biomass developed in the presence of SMP (R1 flora), and biomass developed in reduced level of SMP (R2 flora). We also determined which of these flora produced more refractory SMP. The results showed that R2 flora utilized the synthetic feed more quickly, and produced less refractory organic matter than R0 and R1 flora. The production of more refractory organics by R0 and R1 flora shows that not all the biomass was active. R1 flora degraded the substrates irregularly, suggesting that some microbes were dependent on the metabolic products of those that could utilize the feed components. These results show that production of SMP also depends on the prior substrates and on the ability of the flora to respond to changes in substrate composition.     

Estimation of pairwise relatedness between individuals and characterization of isolation-by-distance processes using dominant genetic markers

A new estimator of the pairwise relatedness coefficient between individuals adapted to dominant genetic markers is developed. This estimator does not assume genotypes to be in Hardy-Weinberg proportions but requires a knowledge of the departure from these proportions (i.e. the inbreeding coefficient). Simulations show that the estimator provides accurate estimates, except for some particular types of individual pairs such as full-sibs, and performs better than a previously developed estimator. When comparing marker-based relatedness estimates with pedigree expectations, a new approach to account for the change of the reference population is developed and shown to perform satisfactorily. Simulations also illustrate that this new relatedness estimator can be used to characterize isolation by distance within populations, leading to essentially unbiased estimates of the neighbourhood size. In this context, the estimator appears fairly robust to moderate errors made on the assumed inbreeding coefficient. The analysis of real data sets suggests that dominant markers (random amplified polymorphic DNA, amplified fragment length polymorphism) may be as valuable as co-dominant markers (microsatellites) in studying microgeographic isolation-by-distance processes. It is argued that the estimators developed should find major applications, notably for conservation biology.     

Orthopoxvirus Genes for Kelch-like Proteins: II. Construction of Cowpox Virus Variants with Targeted Gene Deletions

Integrative plasmids pC, pD, and pG were designed to contain a selective marker beyond the region of homology to virus DNA and to allow construction of recombinant cowpox viruses (CPV) that lack C18L, D11L, or G3L coding for kelch-like proteins. CPV mutants lacking one (C18L, D11L, or G3L), two (D11L/G3L or C18L/D11L), or three (D11L/G3L/C18L, that is, all) kelch-like protein genes of the left variable region of the virus genome were obtained. Impaired reproduction was observed for the triple mutant. Pocks produced by the triple mutant and the original virus differed in size and morphology. In addition, the two CPV variants differed in destructive changes caused in the chorioallantoic membrane of chick embryos.     

Identification and comparison of aerobic and denitrifying polyphosphate-accumulating organisms

Two laboratory-scale sequencing batch reactors (SBRs) were operated for enhanced biological phosphorus removal (EBPR) in alternating anaerobic-aerobic or alternating anaerobic-anoxic modes, respectively. Polyphosphate-accumulating organisms (PAOs) were enriched in the anaerobic-aerobic SBR and denitrifying PAOs (DPAOs) were enriched in the anaerobic-aerobic SBR. Fluorescence in situ hybridization (FISH) demonstrated that the well-known PAO, "Candidatus Accumulibacter phosphatis" was abundant in both SBRs, and post-FISH chemical staining with 4,6-diamidino-2-phenylindol (DAPI) confirmed that they accumulated polyphosphate. When the anaerobic-anoxic SBR enriched for DPAOs was converted to anaerobic-aerobic operation, aerobic uptake of phosphorus by the resident microbial community occurred immediately. However, when the anaerobic-aerobic SBR enriched for PAOs was exposed to one cycle with anoxic rather than aerobic conditions, a 5-h lag period elapsed before phosphorus uptake proceeded. This anoxic phosphorus-uptake lag phase was not observed in the subsequent anaerobic-aerobic cycle. These results demonstrate that the PAOs that dominated the anaerobic-aerobic SBR biomass were the same organisms as the DPAOs enriched under anaerobic-anoxic conditions.     

Synthesis of 3-tert-butylcatechol by an engineered monooxygenase

Recombinant Escherichia coli JM101 was used for the in vivo biocatalytic synthesis of 3-tert-butyl- catechol. The bacterial strain synthesized the laboratory-evolved variant HbpA(T2) of 2-hydroxybiphenyl 3-monooxygenase (HbpA, EC 1.14.13.44) from Pseudomonas azelaica HBP1. The mutant enzyme HbpA(T2) is able to hydroxylate 2-tert-butylphenol to the corresponding catechol, a reaction that is not catalyzed by the wild-type enzyme. The biotransformation was performed in a 3-L bioreactor for 24 h. To mitigate the toxicity of the 2-tert-butylphenol starting material, we applied a limited substrate feed. Continuous in situ product removal with the hydrophobic resin Amberlite XAD-4 was used to separate the product from culture broth. In addition, binding to the resin stabilized the product, which was important because 3-tert-butylcatechol is very labile in aqueous solution. The productivity of the process was 63 mg L(-1) h(-1) so that after 24 h, 3.0 g of 3-tert-butylcatechol were isolated. Down-stream processing consisted of two steps. First, bound 2-tert-butylphenol and 3-tert-butylcatechol were eluted from Amberlite XAD-4 with methanol. Second, the two compounds were separated over neutral aluminum oxide, which selectively binds the produced catechol but not the phenol substrate. The final purity of 3-tert-butylcatechol was greater than 98%.     

Simultaneous nitrification and denitrification using stored substrate (PHB) as the electron donor in an SBR

The potential for PHB (poly-beta-hydroxybutyrate) to serve as the electron donor for effective simultaneous nitrification and denitrification (SND) was investigated in a 2-L sequencing batch reactor (SBR) using a mixed culture and acetate as the organic substrate. During the feast period (i.e., acetate present), heterotrophic respiration activity was high and nitrification was prevented due to the inability of nitrifying bacteria to compete with heterotrophs for oxygen. Once acetate was depleted the oxidation rate of PHB was up to 6 times slower than that of soluble acetate and nitrification could proceed due to the decreased competition for oxygen. The slow nature of PHB degradation meant that it was an effective substrate for SND, as it was oxidised at a similar rate to ammonium and was therefore available for SND throughout the entire aerobic period. The percentage of nitrogen removed via SND increased at lower DO concentrations during the famine period, with up to 78% SND achieved at a DO concentration of 0.5 mg L(-1). However, the increased percentage of SND at a low DO concentration was compromised by a 2-times slower rate of nitrogen removal. A moderate DO concentration of 1 mg L(-1) was optimal for both SND efficiency (61%) and rate (4.4 mmol N x Cmol x(-1) x h(-1)). Electron flux analysis showed that the period of highest SND activity occurred during the first hour of the aerobic famine period, when the specific oxygen uptake rate (SOUR) was highest. It is postulated that a high SOUR due to NH(4) (+) and PHB oxidation decreases oxygen penetration into the floc, creating larger zones for anoxic denitrification. The accumulation of nitrate towards the end of the SND period showed that SND was finally limited by the rate of denitrification. As PHB degradation was found to follow first-order kinetics (df(PHB)/dt = -0.19 x f(PHB)), higher PHB concentrations would be expected to drive SND faster by increasing the availability rate of reducing power and reducing penetration of oxygen into the floc, due to the corresponding increased SOUR. Process control techniques to accumulate higher internal PHB concentrations to improve PHB-driven SND are discussed.