Lucília Domingues

Technological trends, global market, and challenges of bio-ethanol production

Ethanol use as a fuel additive or directly as a fuel source has grown in popularity due to governmental regulations and in some cases economic incentives based on environmental concerns as well as a desire to reduce oil dependency. As a consequence, several countries are interested in developing their internal market for use of this biofuel. Currently, almost all bio-ethanol is produced from grain or sugarcane. However, as this kind of feedstock is essentially food, other efficient and economically viable technologies for ethanol production have been evaluated. This article reviews some current and promising technologies for ethanol production considering aspects related to the raw materials, processes, and engineered strains development. The main producer and consumer nations and future perspectives for the ethanol market are also presented. Finally, technological trends to expand this market are discussed focusing on promising strategies like the use of microalgae and continuous systems with immobilized cells.

Fermentation of lactose to bio-ethanol by yeasts as part of integrated solutions for the valorisation of cheese whey

Cheese whey, the main dairy by-product, is increasingly recognized as a source of many bioactive valuable compounds. Nevertheless, the most abundant component in whey is lactose (ca. 5% w/v), which represents a significant environmental problem. Due to the large lactose surplus generated, its conversion to bio-ethanol has long been considered as a possible solution for whey bioremediation. In this review, fermentation of lactose to ethanol is discussed, focusing on wild lactose-fermenting yeasts, particularly Kluyveromyces marxianus, and recombinant Saccharomyces cerevisiae strains. The early efforts in the screening and characterization of the fermentation properties of wild lactose-consuming yeasts are reviewed. Furthermore, emphasis is given on the latter advances in engineering S. cerevisiae strains for efficient whey-to-ethanol bioprocesses. Examples of industrial implementation are briefly discussed, illustrating the viability of whey-to-ethanol systems. Current developments on strain engineering together with the growing market for biofuels will likely boost the industrial interest in such processes.

Fusion tags for protein solubility, purification and immunogenicity in Escherichia coli: the novel Fh8 system

Proteins are now widely produced in diverse microbial cell factories. The Escherichia coli is still the dominant host for recombinant protein production but, as a bacterial cell, it also has its issues: the aggregation of foreign proteins into insoluble inclusion bodies is perhaps the main limiting factor of the E. coli expression system. Conversely, E. coli benefits of cost, ease of use and scale make it essential to design new approaches directed for improved recombinant protein production in this host cell. With the aid of genetic and protein engineering novel tailored-made strategies can be designed to suit user or process requirements. Gene fusion technology has been widely used for the improvement of soluble protein production and/or purification in E. coli, and for increasing peptide’s immunogenicity as well. New fusion partners are constantly emerging and complementing the traditional solutions, as for instance, the Fh8 fusion tag that has been recently studied and ranked among the best solubility enhancer partners. In this review, we provide an overview of current strategies to improve recombinant protein production in E. coli, including the key factors for successful protein production, highlighting soluble protein production, and a comprehensive summary of the latest available and traditionally used gene fusion technologies. A special emphasis is given to the recently discovered Fh8 fusion system that can be used for soluble protein production, purification, and immunogenicity in E. coli. The number of existing fusion tags will probably increase in the next few years, and efforts should be taken to better understand how fusion tags act in E. coli. This knowledge will undoubtedly drive the development of new tailored-made tools for protein production in this bacterial system.

Optimization of low-cost medium for very high gravity ethanol fermentations by Saccharomyces cerevisiae using statistical experimental designs.

Statistical experimental designs were used to develop a medium based on corn steep liquor (CSL) and other low-cost nutrient sources for high-performance very high gravity (VHG) ethanol fermentations by Saccharomyces cerevisiae. The critical nutrients were initially selected according to a Plackett-Burman design and the optimized medium composition (44.3 g/L CSL; 2.3 g/L urea; 3.8 g/L MgSO₄·7H₂O; 0.03 g/L CuSO₄·5H₂O) for maximum ethanol production by the laboratory strain CEN.PK 113-7D was obtained by response surface methodology, based on a three-level four-factor Box-Behnken design. The optimization process resulted in significantly enhanced final ethanol titre, productivity and yeast viability in batch VHG fermentations (up to 330 g/L glucose) with CEN.PK113-7D and with industrial strain PE-2, which is used for bio-ethanol production in Brazil. Strain PE-2 was able to produce 18.6±0.5% (v/v) ethanol with a corresponding productivity of 2.4±0.1g/L/h. This study provides valuable insights into cost-effective nutritional supplementation of industrial fuel ethanol VHG fermentations.

Wound healing activity of the human antimicrobial peptide LL37

Antimicrobial peptides (AMPs) are part of the innate immune system and are generally defined as cationic, amphipathic peptides, with less than 50 amino acids, including multiple arginine and lysine residues. The human cathelicidin antimicrobial peptide LL37 can be found at different concentrations in many different cells, tissues and body fluids and has a broad spectrum of antimicrobial and immunomodulatory activities. The healing of wound is a complex process that involves different steps: hemostasis, inflammation, remodeling/granulation tissue formation and re-epithelialization. Inflammation and angiogenesis are two fundamental physiological conditions implicated in this process. We have recently developed a new method for the expression and purification of recombinant LL37. In this work, we show that the recombinant peptide P-LL37 with a N-terminus proline preserves its immunophysiological properties in vitro and in vivo. P-LL37 neutralized the activation of macrophages by lipopolysaccharide (LPS). Besides, the peptide induced proliferation, migration and tubule-like structures formation by endothelial cells. Wound healing experiments were performed in dexamethasone-treated mice to study the effect of LL37 on angiogenesis and wound regeneration. The topical application of synthetic and recombinant LL37 increased vascularization and re-epithelialization. Taken together, these results clearly demonstrate that LL37 may have a key role in wound regeneration through vascularization.

Recombinant microbial systems for improved β-galactosidase production and biotechnological applications.

β-Galactosidases (EC 3.2.1.23) constitute a large family of proteins that are known to catalyze both hydrolytic and transgalactosylation reactions. The hydrolytic activity has been applied in the food industry for decades for reducing the lactose content in milk, while the transgalactosylation activity has been used to synthesize galacto-oligosaccharides and galactose containing chemicals in recent years. The main focus of this review is on the expression and production of Aspergillus niger, Kluyveromyces lactis and bacterial β-galactosidases in different microbial hosts. Furthermore, emphasis is given on the reported applications of the recombinant enzymes. Current developments on novel β-galactosidases, derived from newly identified microbial sources or by protein engineering means, together with the use of efficient recombinant microbial production systems are converting this enzyme into a relevant synthetic tool. Thermostable β-galactosidases (cold-adapted or thermophilic) in addition to the growing market for functional foods will likely redouble its industrial interest.

Alcohol production from cheese whey permeate using genetically modified flocculent yeast cells

Alcoholic fermentation of cheese whey permeate was investigated using a recombinant flocculating Saccharomyces cerevisiae, expressing the LAC4 (coding for beta-galactosidase) and LAC12 (coding for lactose permease) genes of Kluyveromyces marxianus enabling for lactose metabolization. Data on yeast fermentation and growth on cheese whey permeate from a Portuguese dairy industry is presented. For cheese whey permeate having a lactose concentration of 50 gL(-1), total lactose consumption was observed with a conversion yield of ethanol close to the expected theoretical value. Using a continuously operating 5.5-L bioreactor, ethanol productivity near 10 g L(-1) h(-1) (corresponding to 0.45 h(-1) dilution rate) was obtained, which raises new perspectives for the economic feasibility of whey alcoholic fermentation. The use of 2-times concentrated cheese whey permeate, corresponding to 100 gL(-1) of lactose concentration, was also considered allowing for obtaining a fermentation product with 5% (w/v) alcohol.

Improving bacterial cellulose for blood vessel replacement: Functionalization with a chimeric protein containing a cellulose-binding module and an adhesion peptide.

Chimeric proteins containing a cellulose-binding module (CBM) and an adhesion peptide (RGD or GRGDY) were produced and used to improve the adhesion of human microvascular endothelial cells (HMEC) to bacterial cellulose (BC). The effect of these proteins on the HMEC-BC interaction was studied. The results obtained demonstrated that recombinant proteins containing adhesion sequences were able to significantly increase the attachment of HMEC to BC surfaces, especially the RGD sequence. The images obtained by scanning electron microscopy showed that the cells on the RGD-treated BC present a more elongated morphology 48h after cell seeding. The results also showed that RGD decreased the in-growth of HMEC cells through the BC and stimulated the early formation of cord-like structures by these endothelial cells. Thus, the use of recombinant proteins containing a CBM domain, with high affinity and specificity for cellulose surfaces allows control of the interaction of this material with cells. CBM may be combined with virtually any biologically active protein for the modification of cellulose-based materials, for in vitro or in vivo applications.

Adaptive evolution of a lactose-consuming Saccharomyces cerevisiae recombinant

ABSTRACT The construction of Saccharomyces cerevisiae strains that ferment lactose has biotechnological interest, particularly for cheese whey fermentation. A flocculent lactose-consuming S. cerevisiae recombinant expressing the LAC12 (lactose permease) and LAC4 (β-galactosidase) genes of Kluyveromyces lactis was constructed previously but showed poor efficiency in lactose fermentation. This strain was therefore subjected to an evolutionary engineering process (serial transfer and dilution in lactose medium), which yielded an evolved recombinant strain that consumed lactose twofold faster, producing 30% more ethanol than the original recombinant. We identified two molecular events that targeted the LAC construct in the evolved strain: a 1,593-bp deletion in the intergenic region (promoter) between LAC4 and LAC12 and a decrease of the plasmid copy number by about 10-fold compared to that in the original recombinant. The results suggest that the intact promoter was unable to mediate the induction of the transcription of LAC4 and LAC12 by lactose in the original recombinant and that the deletion established the transcriptional induction of both genes in the evolved strain. We propose that the tuning of the expression of the heterologous LAC genes in the evolved recombinant was accomplished by the interplay between the decreased copy number of both genes and the different levels of transcriptional induction for LAC4 and LAC12 resulting from the changed promoter structure. Nevertheless, our results do not exclude other possible mutations that may have contributed to the improved lactose fermentation phenotype. This study illustrates the usefulness of simple evolutionary engineering approaches in strain improvement. The evolved strain efficiently fermented threefold-concentrated cheese whey, providing an attractive alternative for the fermentation of lactose-based media.

Production of fermented cheese whey-based beverage using kefir grains as starter culture : evaluation of morphological and microbial variations

Whey valorization concerns have led to recent interest on the production of whey beverage simulating kefir. In this study, the structure and microbiota of Brazilian kefir grains and beverages obtained from milk and whole/deproteinised whey was characterized using microscopy and molecular techniques. The aim was to evaluate its stability and possible shift of probiotic bacteria to the beverages. Fluorescence staining in combination with Confocal Laser Scanning Microscopy showed distribution of yeasts in macro-clusters among the grains matrix essentially composed of polysaccharides (kefiran) and bacteria. Denaturing gradient gel electrophoresis displayed communities included yeast affiliated to Kluyveromyces marxianus, Saccharomyces cerevisiae, Kazachatania unispora, bacteria affiliated to Lactobacillus kefiranofaciens subsp. Kefirgranum, Lactobacillus kefiranofaciens subsp. Kefiranofaciens and an uncultured bacterium also related to the genus Lactobacillus. A steady structure and dominant microbiota, including probiotic bacteria, was detected in the analyzed kefir beverages and grains. This robustness is determinant for future implementation of whey-based kefir beverages.