Strain engineering and process optimization for enhancing the production of a thermostable steryl glucosidase in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Biodiesels produced from transesterification of vegetable oils have a major problem in quality due to the presence of precipitates, which are mostly composed of steryl glucosides (SGs). We have recently described an enzymatic method for the efficient removal of SGs from biodiesel, based on the activity of a thermostable β-glycosidase from Thermococcus litoralis. In the present work, we describe the development of an Escherichia coli-based expression system and a high cell density fermentation process. Strain and process engineering include the assessment of different promoters to drive the expression of a codon-optimized gene, the co-expression of molecular chaperones and the development of a high cell density fermentation process. A 200-fold increase in the production titers was achieved, which directly impacts on the costs of the industrial process for treating biodiesel.     

Enzymatic hydrolysis of steryl glucosides,major contaminants of vegetable oil-derived biodiesel

Biodiesels are mostly produced from lipid transesterification of vegetable oils, including those from soybean, jatropha, palm, rapeseed, sunflower, and others. Unfortunately, transesterification of oil produces various unwanted side products, including steryl glucosides (SG), which precipitate and need to be removed to avoid clogging of filters and engine failures. So far, efficient and cost-effective methods to remove SGs from biodiesel are not available. Here we describe for the first time the identification, characterization and heterologous production of an enzyme capable of hydrolyzing SGs. A synthetic codon-optimized version of the lacS gene from Sulfolobus solfataricus was efficiently expressed and purified from Escherichia coli, and used to treat soybean derived biodiesel containing 100 ppm of SGs. After optimizing different variables, we found that at pH 5.5 and 87 °C, and in the presence of 0.9 % of the emulsifier polyglycerol polyricinoleate, 81 % of the total amount of SGs present in biodiesel were hydrolyzed by the enzyme. This remarkable reduction in SGs suggests a path for the removal of these contaminants from biodiesel on industrial scale using an environmentally friendly enzymatic process.     

Advances in direct transesterification of microalgal biomass for biodiesel production

Microalgae biomass is becoming an interesting raw material to produce biodiesel, where several approaches in the transesterification process have been applied such as different catalysts, different acyl acceptors, incorporation of co-solvents and the different operational conditions. However there are some drawbacks that must be solved before any industrial application could be intended. The main problems are related with the high water content of the biomass (over 80 %) and the several process steps involved in biodiesel production such as: drying, cell disruption, oils extraction, transesterification and biodiesel refining. In comparison to other alternatives, the use of direct transesterification could be a suitable alternative since cell disruption, lipids extraction and transesterification are carried out in one step, with a direct reaction of oil-bearing biomass to biodiesel. This process could be applied even using biomass with high water content, and its efficiency could be improved by the incorporation of promising technologies such as microwave or ultrasonication that can enhance the mass transfer rate between immiscible phases, simultaneously diminishing the reaction time. However, it is still necessary to decrease the costs of these technologies so they can be suitable alternatives in future industrial applications.     

Engineering of microorganisms for the production of biofuels and perspectives based on systems metabolic engineering approaches

The increasing oil price and environmental concerns caused by the use of fossil fuel have renewed our interest in utilizing biomass as a sustainable resource for the production of biofuel. It is however essential to develop high performance microbes that are capable of producing biofuels with very high efficiency in order to compete with the fossil fuel. Recently, the strategies for developing microbial strains by systems metabolic engineering, which can be considered as metabolic engineering integrated with systems biology and synthetic biology, have been developed. Systems metabolic engineering allows successful development of microbes that are capable of producing several different biofuels including bioethanol, biobutanol, alkane, and biodiesel, and even hydrogen. In this review, the approaches employed to develop efficient biofuel producers by metabolic engineering and systems metabolic engineering approaches are reviewed with relevant example cases. It is expected that systems metabolic engineering will be employed as an essential strategy for the development of microbial strains for industrial applications.     

Advanced technologies for improved expression of recombinant proteins in bacteria: perspectives and applications

Prokaryotic expression systems are superior in producing valuable recombinant proteins, enzymes and therapeutic products. Conventional microbial technology is evolving gradually and amalgamated with advanced technologies in order to give rise to improved processes for the production of metabolites, recombinant biopharmaceuticals and industrial enzymes. Recently, several novel approaches have been employed in a bacterial expression platform to improve recombinant protein expression. These approaches involve metabolic engineering, use of strong promoters, novel vector elements such as inducers and enhancers, protein tags, secretion signals, high-throughput devices for cloning and process screening as well as fermentation technologies. Advancement of the novel technologies in E. coli systems led to the production of “difficult to express” complex products including small peptides, antibody fragments, few proteins and full-length aglycosylated monoclonal antibodies in considerable large quantity. Wacker's secretion technologies, Pfenex system, inducers, cell-free systems, strain engineering for post-translational modification, such as disulfide bridging and bacterial N-glycosylation, are still under evaluation for the production of complex proteins and peptides in E. coli in an efficient manner.

This appraisal provides an impression of expression technologies developed in recent times for enhanced production of heterologous proteins in E. coli which are of foremost importance for diverse applications in microbiology and biopharmaceutical production.     

An industrial scale process for the enzymatic removal of steryl glucosides from biodiesel

Background

Biodiesels produced from transesterification of vegetable oils have a major quality problem due to the presence of precipitates, which need to be removed to avoid clogging of filters and engine failures. These precipitates have been reported to be mostly composed of steryl glucosides (SGs), but so far industrial cost-effective methods to remove these compounds are not available. Here we describe a novel method for the efficient removal of SGs from biodiesel, based on the hydrolytic activity of a thermostable β-glycosidase obtained from Thermococcus litoralis.

Results

A steryl glucosidase (SGase) enzyme from T. litoralis was produced and purified from Escherichia coli cultures expressing a synthetic gene, and used to treat soybean-derived biodiesel. Several optimization steps allowed for the selection of optimal reaction conditions to finally provide a simple and efficient process for the removal of SGs from crude biodiesel. The resulting biodiesel displayed filterability properties similar to distilled biodiesel according to the total contamination (TC), the cold soak filtration test (CSFT), filter blocking tendency (FBT), and cold soak filter blocking tendency (CSFBT) tests. The process was successfully scaled up to a 20 ton reactor, confirming its adaptability to industrial settings.

Conclusions

The results presented in this work provide a novel path for the removal of steryl glucosides from biodiesel using a cost-effective, environmentally friendly and scalable enzymatic process, contributing to the adoption of this renewable fuel.

     

微生物与能源的可持续开发

微生物技术在新能源开发领域中有广阔的应用潜力,对能源的可持续发展具有重要的理论和现实意义.简要叙述了生物柴油、燃料酒精、生物制沼气、生物制氢和微生物电池等新能源的原理、优缺点和开发现状,概述了微生物资源在能源领域的应用,指出发掘新的微生物资源或构建工程菌株、明确微生物作用机理、开发新工艺将会是今后研究的重点.     

Cultivation, photobioreactor design and harvesting of microalgae for biodiesel production: A critical review

Microalgae have the ability to mitigate CO₂ emission and produce oil with a high productivity, thereby having the potential for applications in producing the third-generation of biofuels. The key technologies for producing microalgal biofuels include identification of preferable culture conditions for high oil productivity, development of effective and economical microalgae cultivation systems, as well as separation and harvesting of microalgal biomass and oil. This review presents recent advances in microalgal cultivation, photobioreactor design, and harvesting technologies with a focus on microalgal oil (mainly triglycerides) production. The effects of different microalgal metabolisms (i.e., phototrophic, heterotrophic, mixotrophic, and photoheterotrophic growth), cultivation systems (emphasizing the effect of light sources), and biomass harvesting methods (chemical/physical methods) on microalgal biomass and oil production are compared and critically discussed. This review aims to provide useful information to help future development of efficient and commercially viable technology for microalgae-based biodiesel production.     

Superoxide dismutase: an industrial perspective

The application of enzyme technologies to industrial research, development, and manufacturing has become a very important field. Since the production of crude rennet in 1874, several enzymes have been commercialized, and used for therapeutic, supplementary, and other applications. Recent advancements in biotechnology now allow companies to produce safer and less expensive enzymes with enhanced potency and specificity. Antioxidant enzymes are emerging as a new addition to the pool of industrial enzymes and are surpassing all other enzymes in terms of the volume of research and production. In the 1990s, an antioxidant enzyme--superoxide dismutase (SOD)--was introduced into the market. Although the enzyme initially showed great promise in therapeutic applications, it did not perform up to expectations. Consequently, its use was limited to non-drug applications in humans and drug applications in animals. This review summarizes the rise and fall of SOD at the industrial level, the reasons for this, and potential future thrust areas that need to be addressed. The review also focuses on other industrially relevant aspects of SOD such as industrial importance, enzyme engineering, production processes, and process optimization and scale-up.     

Engineering application of anaerobic ammonium oxidation process in wastewater treatment

Anaerobic ammonium oxidation (Anammox), a promising biological nitrogen removal process, has been verified as an efficient, sustainable and cost-effective alternative to conventional nitrification and denitrification processes. To date, more than 110 full-scale anammox plants have been installed and are in operation, treating industrial NH₄ ⁺-rich wastewater worldwide, and anammox-based technologies are flourishing. This review the current state of the art for engineering applications of the anammox process, including various anammox-based technologies, reactor selection and attempts to apply it at different wastewater plants. Process control and implementation for stable performance are discussed as well as some remaining issues concerning engineering application are exposed, including the start-up period, process disturbances, greenhouse gas emissions and especially mainstream anammox applications. Finally, further development of the anammox engineering application is proposed in this review.     

Thermus thermophilus as biological model

Thermus spp is one of the most wide spread genuses of thermophilic bacteria, with isolates found in natural as well as in man-made thermal environments. The high growth rates, cell yields of the cultures, and the constitutive expression of an impressively efficient natural competence apparatus, amongst other properties, make some strains of the genus excellent laboratory models to study the molecular basis of thermophilia. These properties, together with the fact that enzymes and protein complexes from extremophiles are easier to crystallize have led to the development of an ongoing structural biology program dedicated to T. thermophilus HB8, making this organism probably the best so far known from a protein structure point view. Furthermore, the availability of plasmids and up to four thermostable antibiotic selection markers allows its use in physiological studies as a model for ancient bacteria. Regarding biotechnological applications this genus continues to be a source of thermophilic enzymes of great biotechnological interest and, more recently, a tool for the over-expression of thermophilic enzymes or for the selection of thermostable mutants from mesophilic proteins by directed evolution. In this article, we review the properties of this organism as biological model and its biotechnological applications.     

Thermophilic,lignocellulolytic bacteria for ethanol production: current state and perspectives

Lignocellulosic biomass contains a variety of carbohydrates, and their conversion into ethanol by fermentation requires an efficient microbial platform to achieve high yield, productivity, and final titer of ethanol. In recent years, growing attention has been devoted to the development of cellulolytic and saccharolytic thermophilic bacteria for lignocellulosic ethanol production because of their unique properties. First of all, thermophilic bacteria possess unique cellulolytic and hemicellulolytic systems and are considered as potential sources of highly active and thermostable enzymes for efficient biomass hydrolysis. Secondly, thermophilic bacteria ferment a broad range of carbohydrates into ethanol, and some of them display potential for ethanologenic fermentation at high yield. Thirdly, the establishment of the genetic tools for thermophilic bacteria has allowed metabolic engineering, in particular with emphasis on improving ethanol yield, and this facilitates their employment for ethanol production. Finally, different processes for second-generation ethanol production based on thermophilic bacteria have been proposed with the aim to achieve cost-competitive processes. However, thermophilic bacteria exhibit an inherent low tolerance to ethanol and inhibitors in the pretreated biomass, and this is at present the greatest barrier to their industrial application. Further improvement of the properties of thermophilic bacteria, together with the optimization production processes, is equally important for achieving a realistic industrial ethanol production.     

Insights into the Synergistic Biodegradation of Waste Papers Using a Combination of Thermostable Endoglucanase and Cellobiohydrolase from <Emphasis Type="Italic">Chaetomium thermophilum</Emphasis>

Enzymatic hydrolysis is considered an efficient and environmental strategy for the degradation of organic waste materials. Compared to mesophilic cellulases, thermostable cellulases with considerable activity are more advantageous in waste paper hydrolysis, particularly in terms of their participation in synergistic action. In this study, the synergistic effect of two different types of thermostable Chaetomium thermophilum cellulases, the endoglucanase CTendo45 and the cellobiohydrolase CtCel6, on five common kinds of waste papers was investigated. CtCel6 significantly enhanced the bioconversion process, and CTendo45 synergistically increased the degradation, with a maximum degree of synergistic effect of 1.67 when the mass ratio of CTendo45/CtCel6 was 5:3. The synergistic degradation products of each paper material were also determined. Additionally, the activities of CTendo45 and CtCel6 were found to be insensitive to various metals at 2 mM and 10 mM ion concentrations. This study gives an initial insight into a satisfactory synergistic effect of C. thermophilum thermostable cellulases for the hydrolysis of different paper materials, which provides a potential combination of enzymes for industrial applications, including environmentally friendly waste management and cellulosic ethanol production.     

Nanobiotechnology as a novel paradigm for enzyme immobilisation and stabilisation with potential applications in biodiesel production

Nanobiotechnology is emerging as a new frontier of biotechnology. The potential applications of nanobiotechnology in bioenergy and biosensors have encouraged researchers in recent years to investigate new novel nanoscaffolds to build robust nanobiocatalytic systems. Enzymes, mainly hydrolytic class of enzyme, have been extensively immobilised on nanoscaffold support for long-term stabilisation by enhancing thermal, operational and storage catalytic potential. In the present report, novel nanoscaffold variants employed in the recent past for enzyme immobilisation, namely nanoparticles, nanofibres, nanotubes, nanopores, nanosheets and nanocomposites, are discussed in the context of lipase-mediated nanobiocatalysis. These nanocarriers have an inherently large surface area that leads to high enzyme loading and consequently high volumetric enzyme activity. Due to their high tensile strengths, nanoscale materials are often robust and resistant to breakage through mechanical shear in the running reactor making them suitable for multiple reuses. The optimisation of various nanosupports process parameters, such as the enzyme type and selection of suitable immobilisation method may help lead to the development of an efficient enzyme reactor. This might in turn offer a potential platform for exploring other enzymes for the development of stable nanobiocatalytic systems, which could help to address global environmental issues by facilitating the production of green energy. The successful validation of the feasibility of nanobiocatalysis for biodiesel production represents the beginning of a new field of research. The economic hurdles inherent in viably scaling nanobiocatalysts from a lab-scale to industrial biodiesel production are also discussed.     

Enzymatic Processing in Microfluidic Reactors

Abstract

Microreaction technology is an interdisciplinary area of science and engineering. It has attracted the attention of researchers from different fields in the past few years and consequently, several microreactors have been developed. Enzymes are organic catalysts used for the production useful substances in an environmentally friendly way, and have high potential for analytical applications. However, relatively few enzymatic processes have been commercialized because of problems in the stability of enzyme molecule, and the cost and efficiency of the reactions. Thus, there have been demands for innovation in process engineering particularly for enzymatic reactions, and microreaction devices can serve as efficient tools for the development of enzyme processes. In this review, we summarize the recent advances of microchannel reaction technologies and focus our discussion on enzyme microreactors. We discuss the manufacturing process of microreaction devices and the advantages of microreactors compared with the conventional reactors. Fundamental techniques for enzyme microreactors and important applications of this multidisciplinary technology in chemical processing are also included in our topics.     

Esterification using a liquid lipase to remove residual free fatty acids in biodiesel

Lately, the price of liquid formulated lipase enzymes, usable in biodiesel production, has been significantly reduced. This enables one-time use of these enzymes for transesterification, and the process is used industrially. However, the process suffers a drawback by leaving 2−3 % free fatty acids in the crude biodiesel, which reduces the profitability. This article discusses a novel enzymatic FFA esterification reaction utilizing liquid lipase B from Candida antarctica (CALB) along with glycerol at low water concentrations to eliminate the residual FFA. The reaction setup was found able to reduce the free fatty acid concentration to within biodiesel specifications of < 0.25 wt.% FFA. Additionally, two alternative process setups are proposed, which were both found viable through a combination of experiments and simulations, and can be developed into full-scale processes. The resulting two-step enzymatic biodiesel process - transesterification followed by esterification - provides a potential process layout for the industrial production of biodiesel.     

Biofuel production from microalgae as feedstock: current status and potential

Algal biofuel has become an attractive alternative of petroleum-based fuels in the past decade. Microalgae have been proposed as a feedstock to produce biodiesel, since they are capable of mitigating CO₂ emission and accumulating lipids with high productivity. This article is an overview of the updated status of biofuels, especially biodiesel production from microalgae including fundamental research, culture selection and engineering process development; it summarizes research on mathematical and life cycle modeling on algae growth and biomass production; and it updates global efforts of research and development and commercialization attempts. The major challenges are also discussed.     

Lipase from Thermomyces lanuginosus: Uses and prospects as an industrial biocatalyst

The lipase from Thermomyces laguginosus (formerly Humicola laguginosa) (TLL) is a basophilic and noticeably thermostable enzyme, commercially available in both soluble and immobilized form. Although initially oriented toward the food industry, the enzyme has found applications in many different industrial areas, from biodiesel production to fine chemicals (mainly in enantio and regioselective or specific processes). This review intends to show some of the most relevant aspects of the use of this interesting enzyme. After checking the enzyme features, some of the most efficient methods of TLL immobilization will be commented. Finally, the main uses of the enzyme will be revised, with special emphasis in the modification of fats and oils, production of biodiesel, resolution of racemic mixtures, enantioselective hydrolysis of prochiral esters and regioselective process involving sugar preparations. In many instances, TLL has been compared to other lipases, the advantages or disadvantages of the enzyme will be discussed.     

Biodiesel production with microalgae as feedstock: from strains to biodiesel

Due to negative environmental influence and limited availability, petroleum-derived fuels need to be replaced by renewable biofuels. Biodiesel has attracted intensive attention as an important biofuel. Microalgae have numerous advantages for biodiesel production over many terrestrial plants. There are a series of consecutive processes for biodiesel production with microalgae as feedstock, including selection of adequate microalgal strains, mass culture, cell harvesting, oil extraction and transesterification. To reduce the overall production cost, technology development and process optimization are necessary. Genetic engineering also plays an important role in manipulating lipid biosynthesis in microalgae. Many approaches, such as sequestering carbon dioxide from industrial plants for the carbon source, using wastewater for the nutrient supply, and maximizing the values of by-products, have shown a potential for cost reduction. This review provides a brief overview of the process of biodiesel production with microalgae as feedstock. The methods associated with this process (e.g. lipid determination, mass culture, oil extraction) are also compared and discussed.