Screening of white-rot fungi on (14C)lignin-labelled and (14C)whole-labelled wheat straw

Summary 74 Basidiomycetes have been tested for ligninolytic capability on (¹⁴C)lignin-labelled wheat straw. Fifteen strains were selected and rested more accurately for ligninolytic activity and the capacity to degrade wheat straw. The asymptote, inflexion point and degradation rate were determined using a model approach. The fungi exhibited very different responses with respect to lignin biodegradation: high asymptote for Pleurotus ostreatus (77%), low inflexion points for Sporotrichum pulverulentum Nov. (6.1 days) and Pycnoporus spp. (2.7 to 4.7 days) with high and slow degradation rates, respectively (0.91% and 0.45% of ¹⁴CO₂ release/day). Degradation values for (¹⁴C)whole-labelled wheat straw exhibited less variation. Finally, the strains Pleurotus ostreatus, Dichomitus squalens and Bjerkandera adusta showed the highest selectivity of lignin removal.     

Solid substrate fermentation of Monascus purpureus: growth, carbon balance, and consistency analysis

Solid substrate fermentation (SSF) of Monascus purpureus on rice is a promising new technology for obtaining natural pigments. However, before attempts can be made at maximizing pigment yield, all significant macroscopic compounds should be assayed. Here, Monascus purpureus has been grown on rice in batch mode, and the evolution of the main components, biomass, residual rice, O(2), CO(2), ethanol, acetic acid, and pigments, have been followed. This set of data, never previously studied for Monascus SSF, allowed both the performance of a macroscopic elemental balance, which accounted for 83-94% of the initial substrate carbon, and a check of data consistency. Standard consistency analysis showed a significant underestimation of the nitrogen fraction of biomass, but it was unable to discriminate the errors in the carbon balance as a result of the simultaneous presence of two gross errors in the system. A simple stoichiometric model in tandem with consistency analysis explained unaccounted carbon as an underestimation of CO(2) and ethanol. Using the simplified method to estimate ethanol, the macroscopic balance accounted for 87-99% of the initial carbon.     

Epoxide hydrase in Trypanosoma cruzi epimastigotes.

1. Microsomal fractions from Trypanosoma cruzi epimastigotes catalyze the hydration of styrene oxide to styrene glycol. The activity is linear up to 45 min of incubation, is proportional to microsomal protein concentration within certain range, and has an optimum pH of 8.5. 2. Double-reciprocal plots indicate a Km value of 5.3 . 10(-4) M for styrene oxide and a V of 29.6 pmol of styrene glycol formed/min per mg protein at 37 degrees C. 4-Chlorophenyl-2,3-epoxypropyl either (Ki = 2.08 . 10(-4) M) and juvenile hormone I (Ki = 2.7 . 10(-4) M) are competitive inhibitors; whereas, 1-chloro-2,3-epoxypropane is a non-competitive inhibitor. The enzyme is induced about three-fold by 5 mM phenobarbital in the growth medium. 3. The epoxide hydrase is not activated by detergents but rather inhibited by concentrations of Tween-80 and Lubrol as low as 0.025%. 4. Experiments with intact cells indicate that about 3% of [8-14C]styrene oxide penetrates after 90 min of incubation; whereas, over 30% of juvenile hormone I is found intracellularly after the same incubation period. Intracellular styrene oxide is hydrated to styrene glycol to a significant extent and the in vivo hydration is increased by pretreatment with phenobarbital and inhibited upon the addition of 4-chlorophenyl-2,3-epoxypropyl ether. Only a small amount of the intracellular juvenile hormone I is recovered as the corresponding diol ester.     

Data processing for solid substrate cultivation bioreactors

Successful scaling up of Solid Substrate Cultivation (SSC) bioreactors has been hampered by the lack of reliable models that describe such processes satisfactorily. Even though experimental data may be available for model development, data analysis is hindered by system heterogeneity and noisy measurements. This work presents a data processing procedure for periodically agitated SSC fixed bed reactors. The procedure considers several steps. First, all measurements were pre-processed on-line during the cultivation using a low pass fourth order Butterworth digital filter. Then, using this pre-processed data, the average bed temperature, evaporation rate, removed heat, and CO₂ production rate were computed off-line. The variables used to compute the evaporation rate and the removed heat were smoothed off-line with a peak shaving algorithm and a non-delay inducing forward/backward moving average scheme. Variables associated with biomass growth (CO₂ and metabolic heat) are known to evolve slowly. Hence, these were reprocessed with a smoothing procedure in order to diminish the effects of bioreactor heterogeneity. Here, moving average smoothing was applied using a larger window than for other variables, and determined empirically in order to smooth the pre-processed data and extract its real trend. The whole procedure was assessed with data from a 200 kg capacity SSC bioreactor in the cultivation of a filamentous fungus (Gibberella fujikuroi) on wheat bran.     

Metabolic Engineering of Corynebacterium glutamicum for Trehalose Overproduction: Role of the TreYZ Trehalose Biosynthetic Pathway

Trehalose has many potential applications in biotechnology and the food industry due to its protective effect against environmental stress. Our work explores microbiological production methods based on the capacity of Corynebacterium glutamicum to excrete trehalose. We address here raising trehalose productivity through homologous overexpression of maltooligosyltrehalose synthase and the maltooligosyltrehalose trehalohydrolase genes. In addition, heterologous expression of the UDP-glucose pyrophosphorylase gene from Escherichia coli improved the supply of glycogen. Gene expression effects were tested on enzymatic activities and intracellular glycogen content, as well as on accumulated and excreted trehalose. Overexpression of the treY gene and the treY/treZ synthetic operon significantly increased maltooligosyltrehalose synthase activity, the rate-limiting step, and improved the specific productivity and the final titer of trehalose. Furthermore, a strong decrease was noted in glycogen accumulation. Expression of galU/treY and galU/treYZ synthetic operons showed a partial recovery in the intracellular glycogen levels and a significant improvement in both intra- and extracellular trehalose content.     

Ribonucleic acid of Echinococcus granulosus protoscolices

Echinococcus granulosus protoscolices RNA has been characterized in terms of its nucleotide composition, its behavior in sucrose density gradients and methylated albuminkieselguhr columns, its template activity, its biosynthesis kinetics, and the effect of actinomycin D.     

Modeling of yeast metabolism and process dynamics in batch fermentation

Much is known about yeast metabolism and the kinetics of industrial batch fermentation processes. In this study, however, we provide the first tool to evaluate the dynamic interaction that exists between them. A stoichiometric model, using wine fermentation as a case study, was constructed to simulate batch cultures of Saccharomyces cerevisiae. Five differential equations describe the evolution of the main metabolites and biomass in the fermentation tank, while a set of underdetermined linear algebraic equations models the pseudo-steady-state microbial metabolism. Specific links between process variables and the reaction rates of metabolic pathways represent microorganism adaptation to environmental changes in the culture. Adaptation requirements to changes in the environment, optimal growth, and homeostasis were set as the physiological objectives. A linear programming routine was used to define optimal metabolic mass flux distribution at each instant throughout the process. The kinetics of the process arise from the dynamic interaction between the environment and metabolic flux distribution. The model assessed the effect of nitrogen starvation and ethanol toxicity in wine fermentation and it was able to simulate fermentation profiles qualitatively, while experimental fermentation yields were reproduced successfully as well.     

Comparison of aerial and submerged spore properties for Trichoderma harzianum

Abstract Spores produced by aerial mycelium of Trichoderma harzianum PI, a potential biocontrol agent, showed both higher UV-resistance and longer viability after storage than those produced within liquid media ('submerged' spores). Aerial spores were produced in clusters, had a thick outer wall, and few organelles. Trehalose content was significantly lower than in submerged spores. Conversely, submerged spores were mostly collapsed, not clustered and larger than aerial spores. They had many cytoplasmic organelles and a thinner outer wall. These spores were hydrophilic, while aerial ones were highly hydrophobic. On analysis, the latter was related with the presence of a single major low molecular mass protein (< 14 kDa). This protein was nearly absent in extracts from walls of submerged spores but was found in the extracellular medium. An involvement of the outer wall layer in the resting state of T. harzianum spores is proposed.     

A cytosolic FAD-containing enzyme catalyzing cytochrome c reduction in Trypanosoma cruzi. I. Purification and some properties

A cytosolic flavoprotein enzyme for the protozoan, Trypanosoma cruzi, has been purified essentially to homogeneity by DEAE-cellulose and 2',5'-ADP-agarose column chromatography. The native enzyme has a molecular weight of 100,000 +/- 6,000, is composed of two identical subunits of molecular weight 52,000 +/- 1,000, and contains FAD in the ratio of 1 mol of FAD per mol of enzyme subunit. The enzyme is NADPH-dependent and is capable of reducing cytochrome c, ferricyanide, 2,6-dichloroindophenol, and menadione, but not adrenalin. It does not hydroxylate either sodium salicylate or sodium p-hydroxybenzoate, but N-methylaniline and N,N-dimethylaminobenzaldehyde-supported oxidation of NADPH has been demonstrated. Plots of initial velocity against NADPH concentration give hyperbolic curves with Km values of 6.289 X 10(-5) M. The enzyme is clearly different from the microsomal NADPH-cytochrome c reductase in its intracellular distribution, molecular weight, dimeric nature, presence of only FAD, and activity against secondary and tertiary aromatic amines.