C. Casas

Effect of different carbon sources on lipase production by Candida rugosa.

Different carbon sources affecting growth and lipase production in Candida rugosa were studied by using batch cultures on defined medium. Carbohydrates and acids non-related to fats did not induce lipase production. The highest yields of enzyme were obtained with lipids or fatty acids as carbon sources. Tween 80 stimulated lipase biosynthesis and secretion outside the cell. Combinations of two types of substrates, carbohydrates and fatty acids, did not improve lipase production, and in some cases, their consumption was produced in a sequential pattern. Glucose presented a repressing effect on lipase production. Moreover, glucose was found to be effective in stimulating lipase secretion by cells with a high level of cell-bound lipase activity because of their previous growth in oleic acid.

Mechanism of formaldehyde biodegradation by Pseudomonas putida

SummaryFormaldehyde biodegradation by a strain of Pseudomonas putida has been studied. The results indicate that this biodegradation is initiated by a dismutation reaction, yielding as products formic acid and methanol. The degradation of methanol and formic acid begins after exhaustion of formaldehyde in the medium, and presents a diauxic pattern: first formic acid is consumed followed by methanol. Moreover, cell viability, which is affected by the amount of added formaldehyde, has been determined.

Optimization of the heterologous production of a Rhizopus oryzae lipase in Pichia pastoris system using mixed substrates on controlled fed-batch bioprocess.

In this work a systematic study of the influence of methanol set-point and sorbitol feeding rate in fed-batch operation with a Pichia pastoris Mut(s) strain producing Rhizopus oryzae lipase is presented. Different experiments were made at a constant methanol set-point of 0.5, 2 and 4gl(-1), controlled by a predictive algorithm at two different sorbitol feeding rates to assure a constant specific growth rate of 0.01 and 0.02h(-1), by means of a pre-programmed exponential feeding rate strategy. Lipolytic activity, yields, productivity and specific productivity, but also specific growth, consumption and production rates were analyzed showing that the best values were reached when the methanol set-point was 2gl(-1) with a low influence of the constant specific growth rate tested. The sorbitol addition as a co-substrate during the induction phase avoids the severe decrease of the specific production rate obtained when methanol was used as a sole carbon source and it permits to achieve higher ROL production.

Operational aspects of the desulfurization process of energy gases mimics in biotrickling filters

Biological removal of reduced sulfur compounds in energy-rich gases is an increasingly adopted alternative to conventional physicochemical processes, because of economical and environmental benefits. A lab-scale biotrickling filter reactor for the treatment of high-H(2)S-loaded gases was developed and previously proven to effectively treat H(2)S concentrations up to 12,000 ppm(v) at gas contact times between 167 and 180 s. In the present work, a detailed study on selected operational aspects affecting this system was carried out with the objective to optimize performance. The start-up phase was studied at an inlet H(2)S concentration of 1000 ppm(v) (loading of 28 g H(2)S m(-3) h(-1)) and inoculation with sludge from a municipal wastewater treatment plant. After reactor startup, the inlet H(2)S concentration was doubled and the influence of different key process parameters was tested. Results showed that there was a significant reduction of the removal efficiency at gas contact times below 120 s. Also, mass transfer was found to be the main factor limiting H(2)S elimination, whereas performance was not influenced by the bacterial colonization of the packed column after the initial startup. The effect of gas supply shutdowns for up to 5 days was shown to be irrelevant on process performance if the trickling liquid recirculation was kept on. Also, the trickling liquid velocity was investigated and found to influence sulfate production through a better use of the supplied dissolved oxygen. Finally, short-term pH changes revealed that the system was quite insensitive to a pH drop, but was markedly affected by a pH increase, affecting both the biological activity and the removal of H(2)S. Altogether, the results presented and discussed herein provide new insight and operational data on H(2)S removal from energy gases in biotrickling filters.

Immobilized cells: behaviour of carrageenan entrapped yeast during continuous ethanol fermentation

SummaryData of cell concentration, viability and microscopic observation of cell distribution inside carrageenan immobilized yeast beads are reported. Results were obtained from a continuous packed-bed reactor performing alcoholic fermentation and the main observations made on cell activity are in agreement with the fermentation profiles inside the fermenter.

IMPROVING LIPASE PRODUCTION FROM CANDIDA RUGOSA BY A BIOCHEMICAL ENGINEERING APPROACH

It has been tested that the use of oleic acid as sole carbon source and as inducer of the production has an important effect in the lipase production by Candida rugosa under aerobic conditions. A simple structured mathematical model coupled with a methodology to estimate biomass, specific growth rate and substrate was developed and applied to the production of Candida rugosa lipase in batch, fed-batch and continuous operation to obtain a reproducible product. The best operation mode tested was a controlled specific growth rate fed-batch with a 10-fold increase in productivity related to batch operation. Downstream of the culture broth has demonstrated that the ratio between the different isoenzymes presented can be modulated by the selection of the operational strategy and this ratio is quite different comparing with commercial lipases. Thus, their catalytic properties in front of chiral reactions could be different.

Analysis of Nutritional Factors and Physical Conditions Affecting Growth and Monoclonal Antibody Production of the Hybridoma KB-26.5 Cell Line

The effect of medium composition and physical conditions on the growth pattern and monoclonal antibody production of the hybridoma cell line KB‐26.5 has been studied in batch and fed‐batch cultures. Different aspects have been analyzed both individually and in combination, as it is expected that not only one component plays a predominant role in this process but a combination of a number of them. Modification of the concentrations of glucose and glutamine, supplementation of the medium with vitamins and amino acids, influence of lactate and ammonium accumulation, and pulse addition of fetal calf serum have been studied in detail, contributing to an improvement in the cell growth and IgG3 production. Finally, the combined effect of all these factors in a fed‐batch culture leads clearly to a major improvement in the cultures, with a 10‐fold increase in the final monoclonal antibody concentration with respect to nonoptimized batch experiments.

Determination of the intrinsic fermentation kinetics of Saccharomyces cerevisiae cells immobilized in Ca-alginate beads and observations on their growth

Continuous fermentation experiments in a well-stirred fermentor with Saccharomyces cerevisiae cells immobilized in Ca-alginate beads of small diameter (approx. 1 mm) have been performed in order to discover their intrinsic fermentation kinetics, and compare them to the fermentation kinetics for free cells, by fitting both sets of results to the same model. The results show similar kinetic parameters for free and immobilized cells. The changes in cell concentration inside the beads and microscopical observations of transverse sections throughout the experiments, allowed discernment of two different scenarios of cell growth inside the beads: low cell density and fully developed growth.

Secretion of the sweet-tasting protein thaumatin by recombinant strains of Aspergillus niger var. awamori

A recombinant form of the sweet-tasting protein thaumatin has been produced in the filamentous fungus Aspergillus niger var. awamori. Expression cassettes containing a synthetic gene encoding thaumatin II were prepared and used to transform Aspergillus niger var. awamori strain NRRL312. Several fungal strains capable of synthesizing and secreting thaumatin into the culture medium were generated, and their production capabilities were determined, first in shake flasks and later in a laboratory fermentor. We report the expression and secretion of thaumatin in concentrations of 5–7 mg/l. This recombinant thaumatin is sweet.

Study of the drop size frequencies in a microbial growth system with an aqueous-organic culture medium: lipase production from Candida rugosa

The influence of the drop size of oleic acid on extracellular lipase production by Candida rugosa was studied. The specific interfacial area was determined under different experimental conditions and the drop populations obtained were compared on the basis of the Kruskal-Wallis statistical test. Lipase production was found to increase with increasing drop size of the disperse phase, i.e. when the microorganism grew at a low stirring rate (300 rpm). Enzyme production in two bioreactors of different volume, viz. Biostat E (5 l) and Bioflo III (1 l), was also compared. The two bioreactors were found to yield similar enzyme activities provided the same initial oleic acid drop size was used. This allows the establishment of a specific criterion for adaptation to a different scale or bioreactor, and for the mathematical modelling of the process