Ana M.R.B. Xavier

Concomitant substrate and product inhibition kinetics in lactic acid production

The influence of glucose inhibition on growth and lactic acid production kinetics of Lactobacillus delbrueckii NRRL B445 was studied in batch cultures. Continuous experiments (CSTR) were also carried out. A model for substrate consumption, cell growth, and lactic acid production considering both substrate and product inhibition is proposed; this model fits experimental data better than previously described models, since these have only considered low substrate concentrations. The kinetics of continuous culture can be also described adequately by the model developed from the batch tests.

Optimization and Modeling of Laccase Production by Trametes versicolor in a Bioreactor Using Statistical Experimental Design

Experimental design and response surface methodologies were applied to optimize laccase production by Trametes versicolor in a bioreactor. The effects of three factors, initial glucose concentration (0 and 9 g/L), agitation (100 and 180 rpm), and pH (3.0 and 5.0), were evaluated to identify the significant effects and its interactions in the laccase production. The pH of the medium was found to be the most important factor, followed by initial glucose concentration and the interaction of both factors. Agitation did not seem to play an important role in laccase production, nor did the interaction agitation x medium pH and agitation x initial glucose concentration. Response surface analysis showed that an initial glucose concentration of 11 g/L and pH controlled at 5.2 were the optimal conditions for laccase production by T. versicolor. Under these conditions, the predicted value for laccase activity was >10,000 U/L, which is in good agreement with the laccase activity obtained experimentally (11,403 U/L). In addition, a mathematical model for the bioprocess was developed. It is shown that it provides a good description of the experimental profile observed, and that it is capable of predicting biomass growth based on secondary process variables.

Elucidation of the mechanism of lactic acid growth inhibition and production in batch cultures of Lactobacillus rhamnosus

Abstract Batch cultures of Lactobacillus rhamnosus were carried out at different pH values in order to study the limitation of growth and lactic acid production by the hydrogen ion, non-dissociated lactic acid and internal lactate concentrations. The effect of pH between 5 and 6.8 was studied at non-limiting concentrations of glucose; this is more significant for the lactic acid fermentation rate than for the maximum specific growth rate, as shown by the incomplete substrate consumption at lower values of medium pH and by the constant maximum cell mass obtained within the range of pH values studied. To check whether these results were a direct consequence of the different concentrations of the non-dissociated form of lactic acid at different external pH values, specific growth rates and lactic acid productions rates were calculated for each external pH value. The same specific growth rates were observed at the same non-dissociated lactic acid concentrations only at pH values of 5 and 5.5. For higher values of pH (pH > 6) the specific growth rate falls to zero as the non-dissociated lactic acid concentration decreases. This shows that generalisations made from studies performed within very narrow ranges of pH are not valid and that the non-dissociated form of lactic acid is not the only inhibiting species. The internal pH was measured experimentally for each external pH value in order to calculate the internal lactate ion concentration. This form is described to be the inhibitory one. The results obtained confirmed that the specific growth rate reached zero at approximately the same lactate concentration for all the pH values studied.

Second-generation bioethanol from eucalypt sulphite spent liquor

The spent liquor from acidic sulphite pulping of Eucalyptus globulus (HSSL) is the side product from sulphite pulp production and besides sulphonated lignin contains sugars from degraded hemicelluloses, mainly pentoses. Pichia stipitis fermentation of these sugars for bioethanol production was the primary goal of this work. The increasing of HSSL proportion in fermentation media affected negatively the ethanol yield. Thus with 20% of HSSL (v/v) attained maximum ethanol yield was 0.15 g of ethanol by g of sugar consumed (g(e) g(s)(-1)) and with 60% (v/v) only 0.08 g(e) g(s)(-1). Biological removal of acetic acid from HSSL improved fermentation though the complete removal of acetic acid and polyphenolics (including sulphonated species) by treatment with ion-exchange resins was required for highly successful bioethanol production. Accordingly, the fermentative metabolic pathway of P. stipitis has been promoted allowing fair ethanol productivity and yield (Yp/s = 0.49 g(e) g(s)(-1)) at relatively low maximum of cell growth rate (micro(max) = 0.21 h(-1)).

Degradation of biphenyl lignin model compounds by laccase of Trametes versicolor in the presence of 1-hydroxybenzotriazole and heteropolyanion [SiW11VO40]5−

Abstract A series of phenolic/non-phenolic biphenyl model compounds mimicking 5-5′ type “condensed” lignin substructures were subjected to the oxidation with laccase of Trametes versicolor in the presence of 1-hydroxybenzotriazole (HBT) or [SiW 11 VO 40 ] 5− (SiW 11 V) as mediators. Phenolic models suffered a significant degradation in both the laccase-mediator systems (LMS), which was more pronounced, however, in the case of SiW 11 V-mediated oxidation. This result was explained, at least partially, by HBT decomposition and by the increased extent of competing radical coupling reactions of phenolic models in the HBT–laccase reaction system. The non-phenolic biphenyl model was non-reactive in the presence of SiW 11 V and degraded substantially in the presence of HBT. The main degradation pathways of lignin model compounds were deduced based on the analysis of the detected oxidation products.

Enzymatic saccharification and bioethanol production from Cynara cardunculus pretreated by steam explosion.

The correct choice of the specific lignocellulosic biomass pretreatment allows obtaining high biomass conversions for biorefinery implementations and cellulosic bioethanol production from renewable resources. Cynara cardunculus (cardoon) pretreated by steam explosion (SE) was involved in second-generation bioethanol production using separate hydrolysis and fermentation (SHF) or simultaneous saccharification and fermentation (SSF) processes. Steam explosion pretreatment led to partial solubilisation of hemicelluloses and increased the accessibility of residual polysaccharides towards enzymatic hydrolysis revealing 64% of sugars yield against 11% from untreated plant material. Alkaline extraction after SE pretreatment of cardoon (CSEOH) promoted partial removal of degraded lignin, tannins, extractives and hemicelluloses thus allowing to double glucose concentration upon saccharification step. Bioethanol fermentation in SSF mode was faster than SHF process providing the best results: ethanol concentration 18.7 g L(-1), fermentation efficiency of 66.6% and a yield of 26.6g ethanol/100 g CSEOH or 10.1 g ethanol/100 g untreated cardoon.

Biological treatment of eucalypt spent sulphite liquors: A way to boost the production of second generation bioethanol

The fermentation of reducing sugars from hardwood (eucalypt) spent sulphite liquor (HSSL) into ethanol by Pichia (Scheffersomyces) stipitis is hindered by concomitant inhibitors of microbial metabolism. The conditions for the HSSL biological treatment step by Paecilomyces variotii were evaluated and optimised. Two different strategies of reactor operation were compared using single batch (B) and sequential batch reactor (SBR). Biological treatment of HSSL in the SBR revealed the best results with respect to the removal of microbial inhibitors. Also, most of inhibitory compounds, acetic acid, gallic acid, pyrogallol, amongst others, were removed from HSSL by P. variotii before the ethanol fermentation. The bio-detoxified HSSL was subjected to a successful fermentation by P. stipitis, attaining a maximum ethanol concentration of 2.4 g L(-1) with a yield of 0.24 g ethanol g sugars(-1).

Tangential flow filtration for continuous cell recycle culture of acidogenic bacteria

Abstract The operation of cell recycle systems by membrane filtration is discussed, stressing the importance of the mutual interaction between the biological and the physical characteristics of the fermentation process. Broth circulation flow rate, dilution rate, and cell bleed rate are identified as being the most important process variables. Their effects on process performance and criteria for its adequate selection are analysed. Fermentation results for different acidogenic microorganisms are presented and the kinetic modelling of high cell density recycling systems is discussed.

Bacterial cellulose as carrier for immobilization of laccase: Optimization and characterization

Bacterial cellulose (BC) has attracted attention as a new functional material due to its excellent mechanical strength, tridimensional nanostructure, high purity, and increased water absorption, compared to plant cellulose. In this work, commercial laccase was immobilized on BC and the influence of enzyme concentration, contact time, and pH was optimized toward the recovery activity of immobilized laccase. This optimization was carried out using a 33 experimental design and response surface methodology. Enzyme concentration played a critical role in laccase immobilization. Under optimized conditions (0.15 μL L−1 of enzyme concentration, 4.8 h of contact time, pH 5.4), the predicted and experimental response were equal to 47.88 and 49.30%, respectively. The thermal stability of the immobilized laccase was found to increase notably at 60 and 70°C presenting stabilization factor equal to 1.79 and 2.11, respectively. The immobilized laccase showed high operational stability, since it retained 86% of its initial activity after seven consecutive biocatalytic cycles of reaction with 2,2′‐azinobis‐(3‐ethylbenzothiazoline‐6‐sulfonic acid). Kinetic studies showed that the values of Michaelis–Menten constant and maximum reaction rate decreased upon immobilization (9.9‐ and 1.6‐fold, respectively). Globally, the use of immobilized laccase on BC offers an interesting tool for industrial biocatalytic applications.

Adaptation of Scheffersomyces stipitis to hardwood spent sulfite liquor by evolutionary engineering

BackgroundHardwood spent sulfite liquor (HSSL) is a by-product of acid sulfite pulping process that is rich in xylose, a monosaccharide that can be fermented to ethanol by Scheffersomyces stipitis. However, HSSL also contains acetic acid and lignosulfonates that are inhibitory compounds of yeast growth. The main objective of this study was the use of an evolutionary engineering strategy to obtain variants of S. stipitis with increased tolerance to HSSL inhibitors while maintaining the ability to ferment xylose to ethanol.ResultsA continuous reactor with gradually increasing HSSL concentrations, from 20% to 60% (v/v), was operated for 382 generations. From the final obtained population (POP), a stable clone (C4) was isolated and characterized in 60% undetoxified HSSL. C4 isolate was then compared with both the parental strain (PAR) and POP. Both POP and C4 were able to grow in 60% undetoxified HSSL, with a higher capability to withstand HSSL inhibitors than PAR. Higher substrate uptake rates, 7% higher ethanol efficiency and improved ethanol yield were obtained using C4.ConclusionS. stipitis was successfully adapted to 60% (v/v) undetoxified eucalyptus HSSL. A stable isolate, C4, with an improved performance in undetoxified HSSL compared to PAR was successfully obtained from POP. Owing to its improved tolerance to inhibitors, C4 may represent a major advantage for the production of bioethanol using HSSL as substrate.