A new dawn for industrial photosynthesis

Several emerging technologies are aiming to meet renewable fuel standards, mitigate greenhouse gas emissions, and provide viable alternatives to fossil fuels. Direct conversion of solar energy into fungible liquid fuel is a particularly attractive option, though conversion of that energy on an industrial scale depends on the efficiency of its capture and conversion. Large-scale programs have been undertaken in the recent past that used solar energy to grow innately oil-producing algae for biomass processing to biodiesel fuel. These efforts were ultimately deemed to be uneconomical because the costs of culturing, harvesting, and processing of algal biomass were not balanced by the process efficiencies for solar photon capture and conversion. This analysis addresses solar capture and conversion efficiencies and introduces a unique systems approach, enabled by advances in strain engineering, photobioreactor design, and a process that contradicts prejudicial opinions about the viability of industrial photosynthesis. We calculate efficiencies for this direct, continuous solar process based on common boundary conditions, empirical measurements and validated assumptions wherein genetically engineered cyanobacteria convert industrially sourced, high-concentration CO₂ into secreted, fungible hydrocarbon products in a continuous process. These innovations are projected to operate at areal productivities far exceeding those based on accumulation and refining of plant or algal biomass or on prior assumptions of photosynthetic productivity. This concept, currently enabled for production of ethanol and alkane diesel fuel molecules, and operating at pilot scale, establishes a new paradigm for high productivity manufacturing of nonfossil-derived fuels and chemicals.     

Protein crystallization in stirred systems—scale‐up via the maximum local energy dissipation

Macromolecular bioproducts like therapeutic proteins have usually been crystallized with µL‐scale vapor diffusion experiments for structure determination by X‐ray diffraction. Little systematic know‐how exists for technical‐scale protein crystallization in stirred vessels. In this study, the Fab‐fragment of the therapeutic antibody Canakinumab was successfully crystallized in a stirred‐tank reactor on a 6 mL‐scale. A four times faster onset of crystallization of the Fab‐fragment was observed compared to the non‐agitated 10 µL‐scale. Further studies on a liter‐scale with lysozyme confirmed this effect. A 10 times faster onset of crystallization was observed in this case at an optimum stirrer speed. Commonly suggested scale‐up criteria (i.e., minimum stirrer speed to keep the protein crystals in suspension or constant impeller tip speed) were shown not to be successful. Therefore, the criterion of constant maximum local energy dissipation was applied for scale‐up of the stirred crystallization process for the first time. The maximum local energy dissipation was estimated by measuring the drop size distribution of an oil/surfactant/water emulsion in stirred‐tank reactors on a 6 mL‐, 100 mL‐, and 1 L‐scale. A comparable crystallization behavior was achieved in all stirred‐tank reactors when the maximum local energy dissipation was kept constant for scale‐up. A maximum local energy dissipation of 2.2 W kg^?1 was identified to be the optimum for lysozyme crystallization at all scales under study. Biotechnol. Bioeng. 2013; 110: 1956–1963. © 2013 Wiley Periodicals, Inc.     

Integration of microbial kinetics and fluid dynamics toward model‐driven scale‐up of industrial bioprocesses

Scale‐up of bioprocesses is hampered by open questions, mostly related to poor mixing and mass transfer limitations. Concentration gradients of substrate, carbon dioxide, and oxygen in time and space, especially in large‐scale high‐cell density fed‐batch processes, are likely induced as the mixing time of the fermentor is usually longer than the relevant cellular reaction time. Cells in the fermentor are therefore repeatedly exposed to dynamic environments or perturbations. As a consequence, the heterogeneity in industrial practices often decreases either yield, titer, or productivity, or combinations thereof and increases by‐product formation as compared to well‐mixed small‐scale bioreactors, which is summarized as scale‐up effects. Identification of response mechanisms of the microorganism to various external perturbations is of great importance for pinpointing metabolic bottlenecks and targets for metabolic engineering. In this review, pulse response experimentation is proposed as an ideal way of obtaining kinetic information in combination with scale‐down approaches for in‐depth understanding of dynamic response mechanisms. As an emerging tool, computational fluid dynamics is able to draw a holistic picture of the fluid flow and concentration fields in the fermentor and finds its use in the optimization of fermentor design and process strategy. In the future, directed strain improvement and fermentor redesign are expected to largely depend on models, in which both microbial kinetics and fluid dynamics are thoroughly integrated.     

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.     

Monolith enzymatic microreactor at the frontier of glycomic toward a new route for the production of bioactive oligosaccharides

Recent research in the area of bioactive carbohydrates has shown the efficiency of oligosaccharides as signal molecules in a lot of biological activities. Newly observed functions of oligosaccharides and their abilities to act as specific regulatory molecules on various organisms have been more and more described. A successful development of these bioactive molecules in future needs efficient processes for specific oligosaccharides production. To exploit them for putative industrial scale up processes, two main strategies are currently investigated: the synthesis (chemical or bioconversion processes) and the polysaccharide cleavage (chemical, physical or biological processes). Nevertheless, if new manufacturing biotechnologies have considerably increased the development of these functional molecules, the main drawback limiting their biological applications is the complexity to engender specific glycosidic structures for specific activities. In the recent years, new enzymatic reactors have been developed, allowing the automatic synthesis of oligosaccharide structures. This review focuses on the knowledge in the area of bioactive oligosaccharides and gives the main processes employed to generate them for industrial applications with challenges of monolith microreactors.     

Evaluating scaling of capillary photo-biofilm reactors for high cell density cultivation of mixed trophies artificial microbial consortia

Capillary biofilm reactors (CBRs) are attractive for growing photoautotrophic bacteria as they allow high cell-density cultivation. Here, we evaluated the CBR system's suitability to grow an artificial consortium composed of Synechocystis sp. PCC 6803 and Pseudomonas sp. VBL120. The impact of reactor material, flow rate, pH, O₂, and medium composition on biomass development and long-term biofilm stability at different reactor scales was studied. Silicone was superior over other materials like glass or PVC due to its excellent O₂ permeability. High flow rates of 520 μL min⁻¹ prevented biofilm sloughing in 1 m capillary reactors, leading to a 54% higher biomass dry weight combined with the lowest O₂ concentration inside the reactor compared to standard operating conditions. Further increase in reactor length to 5 m revealed a limitation in trace elements. Increasing trace elements by a factor of five allowed for complete surface coverage with a biomass dry weight of 36.8 g m⁻² and, thus, a successful CBR scale-up by a factor of 25. Practical application : Cyanobacteria use light energy to upgrade CO₂, thereby holding the potential for carbon-neutral production processes. One of the persisting challenges is low cell density due to light limitations and O₂ accumulation often occurring in established flat panel or tubular photobioreactors. Compared to planktonic cultures, much higher cell densities (factor 10 to 100) can be obtained in cyanobacterial biofilms. The capillary biofilm reactor (CBR) offers good growth conditions for cyanobacterial biofilms, but its applicability has been shown only on the laboratory scale. Here, a first scale-up study based on sizing up was performed, testing the feasibility of this system for large-scale applications. We demonstrate that by optimizing nutrient supply and flow conditions, the system could be enlarged by factor 25 by enhancing the length of the reactor. This reactor concept, combined with cyanobacterial biofilms and numbering up, holds the potential to be applied as a flexible, carbon-neutral production platform for value-added compounds.     

WAVETM反应器和搅拌瓶中微载体球转球放大培养的比较

目的:哺乳动物细胞目前已广泛用于生物工程药物如单抗和疫苗的生产.而用于贴壁细胞规模化培养的微载体,也应时应需得以开发并应用于生物制药.贴壁细胞微载体培养在搅拌罐和WAVETM反应器中都能进行.而如要进行进一步的放大培养,球转球工艺不可或缺.为了发展球转球这一新的放大技术,以及考量WAVETM反应器这种新型大规模培养设备的应用性,大量的细胞培养和球转球实验在WAVETM反应器和搅拌瓶中进行.收集到的数据得以分析比较.方法:将Vero细胞分别接入WAVETM反应器和搅拌瓶中用微载体Cytodex 1进行培养.适当补充营养并控制温度、pH等培养条件使细胞增殖.长满微载体的细胞用清洗、消化等球转球工艺的一系列步骤而分离,并放大接种到新的培养体系.球转球工艺的有效性通过记录并统计分析细胞消化分离的回收率,以及细胞重新接种生长的存活力来评估.结果:统计学分析比较WAVETM反应器和搅拌瓶中得到的细胞分离回收率分别是67.56％和39.39％,数理统计P值小于0.0003;细胞重新接种存活率分别是95.17％和78.45％,P值等于0.0107.结论:在WAVETM反应器中进行的球转球放大工艺,其总体表现和有效性远高于在搅拌瓶中得到的结果.在WAVETM反应器中培养的Vero细胞有很好的细胞状态,作为种子链和生产用罐相比搅拌型反应罐均有很大的优越性.     

Developing Bacillus spp. as a cell factory for production of microbial enzymes and industrially important biochemicals in the context of systems and synthetic biology

Increasing concerns over limited petroleum resources and associated environmental problems are motivating the development of efficient cell factories to produce chemicals, fuels, and materials from renewable resources in an environmentally sustainable economical manner. Bacillus spp., the best characterized Gram-positive bacteria, possesses unique advantages as a host for producing microbial enzymes and industrially important biochemicals. With appropriate modifications to heterologous protein expression and metabolic engineering, Bacillus species are favorable industrial candidates for efficiently converting renewable resources to microbial enzymes, fine chemicals, bulk chemicals, and fuels. Here, we summarize the recent advances in developing Bacillus spp. as a cell factory. We review the available genetic tools, engineering strategies, genome sequence, genome-scale structure models, proteome, and secretion pathways, and we list successful examples of enzymes and industrially important biochemicals produced by Bacillus spp. Furthermore, we highlight the limitations and challenges in developing Bacillus spp. as a robust and efficient production host, and we discuss in the context of systems and synthetic biology the emerging opportunities and future research prospects in developing Bacillus spp. as a microbial cell factory.     

A review of biological delignification and detoxification methods for lignocellulosic bioethanol production

Future biorefineries will integrate biomass conversion processes to produce fuels, power, heat and value-added chemicals. Due to its low price and wide distribution, lignocellulosic biomass is expected to play an important role toward this goal. Regarding renewable biofuel production, bioethanol from lignocellulosic feedstocks is considered the most feasible option for fossil fuels replacement since these raw materials do not compete with food or feed crops. In the overall process, lignin, the natural barrier of the lignocellulosic biomass, represents an important limiting factor in biomass digestibility. In order to reduce the recalcitrant structure of lignocellulose, biological pretreatments have been promoted as sustainable and environmentally friendly alternatives to traditional physico-chemical technologies, which are expensive and pollute the environment. These approaches include the use of diverse white-rot fungi and/or ligninolytic enzymes, which disrupt lignin polymers and facilitate the bioconversion of the sugar fraction into ethanol. As there is still no suitable biological pretreatment technology ready to scale up in an industrial context, white-rot fungi and/or ligninolytic enzymes have also been proposed to overcome, in a separated or in situ biodetoxification step, the effect of the inhibitors produced by non-biological pretreatments. The present work reviews the latest studies regarding the application of different microorganisms or enzymes as useful and environmentally friendly delignification and detoxification technologies for lignocellulosic biofuel production. This review also points out the main challenges and possible ways to make these technologies a reality for the bioethanol industry.     

Changing the way we think about global change research: scaling up in experimental ecosystem science

Scaling is a naturally iterative and bi‐directional component of problem solving in ecology and in climate science. Ecosystems and climate systems are unquestionably the sum of all their parts, to the smallest imaginable scale, in genomic processes or in the laws of fluid dynamics. However, in the process of scaling‐up, for practical purposes thewhole usually has to be construed as a good deal less than this. This essay demonstrates how controlled large‐scale experiments can be used to deduce key mechanisms and thereby reduce much of the detail needed for the process of scaling‐up. Collection of the relevant experimental evidence depends on controlling the environment and complexity of experiments, and on applications of technologies that report on, and integrate, small‐scale processes. As the role of biological feedbacks in the behavior of climate systems is better appreciated, so the need grows for experimentally based understanding of ecosystem processes. We argue that we cannot continue as we are doing, simply observing the progress of the greenhouse gas‐driven experiment in global change, and modeling its future outcomes. We have to change the way we think about climate system and ecosystem science, and in the process move to experimental modes at larger scales than previously thought achievable.     

Integration of microalgae cultivation with industrial waste remediation for biofuel and bioenergy production: opportunities and limitations

There is currently a renewed interest in developing microalgae as a source of renewable energy and fuel. Microalgae hold great potential as a source of biomass for the production of energy and fungible liquid transportation fuels. However, the technologies required for large-scale cultivation, processing, and conversion of microalgal biomass to energy products are underdeveloped. Microalgae offer several advantages over traditional 'first-generation' biofuels crops like corn: these include superior biomass productivity, the ability to grow on poor-quality land unsuitable for agriculture, and the potential for sustainable growth by extracting macro- and micronutrients from wastewater and industrial flue-stack emissions. Integrating microalgal cultivation with municipal wastewater treatment and industrial CO(2) emissions from coal-fired power plants is a potential strategy to produce large quantities of biomass, and represents an opportunity to develop, test, and optimize the necessary technologies to make microalgal biofuels more cost-effective and efficient. However, many constraints on the eventual deployment of this technology must be taken into consideration and mitigating strategies developed before large scale microalgal cultivation can become a reality. As a strategy for CO(2) biomitigation from industrial point source emitters, microalgal cultivation can be limited by the availability of land, light, and other nutrients like N and P. Effective removal of N and P from municipal wastewater is limited by the processing capacity of available microalgal cultivation systems. Strategies to mitigate against the constraints are discussed.     

Livestock waste-to-bioenergy generation opportunities

The use of biological and thermochemical conversion (TCC) technologies in livestock waste-to-bioenergy treatments can provide livestock operators with multiple value-added, renewable energy products. These products can meet heating and power needs or serve as transportation fuels. The primary objective of this work is to present established and emerging energy conversion opportunities that can transform the treatment of livestock waste from a liability to a profit center. While biological production of methanol and hydrogen are in early research stages, anaerobic digestion is an established method of generating between 0.1 to 1.3m3m(-3)d(-1) of methane-rich biogas. The TCC processes of pyrolysis, direct liquefaction, and gasification can convert waste into gaseous fuels, combustible oils, and charcoal. Integration of biological and thermal-based conversion technologies in a farm-scale hybrid design by combining an algal CO2-fixation treatment requiring less than 27,000m2 of treatment area with the energy recovery component of wet gasification can drastically reduce CO2 emissions and efficiently recycle nutrients. These designs have the potential to make future large scale confined animal feeding operations sustainable and environmentally benign while generating on-farm renewable energy.     

Zonal rate model for axial and radial flow membrane chromatography,part II: Model‐based scale‐up

Membrane chromatography (MC) systems are finding increasing use in downstream processing trains for therapeutic proteins due to the unique mass‐transfer characteristics they provide. As a result, there is increased need for model‐based methods to scale‐up MC units using data collected on a scaled‐down unit. Here, a strategy is presented for MC unit scale‐up using the zonal rate model (ZRM). The ZRM partitions an MC unit into virtual flow zones to account for deviations from ideal plug‐flow behavior. To permit scale‐up, it is first configured for the specific device geometry and flow profiles within the scaled‐down unit so as to achieve decoupling of flow and binding related non‐idealities. The ZRM is then configured for the preparative‐scale unit, which typically utilizes markedly different flow manifolds and membrane architecture. Breakthrough is first analyzed in both units under non‐binding conditions using an inexpensive tracer to independently determine unit geometry related parameters of the ZRM. Binding related parameters are then determined from breakthrough data on the scaled‐down MC capsule to minimize sample requirements. Model‐based scale‐up may then be performed to predict band broadening and breakthrough curves on the preparative‐scale unit. Here, the approach is shown to be valid when the Pall XT140 and XT5 capsules serve as the preparative and scaled‐down units, respectively. In this case, scale‐up is facilitated by our finding that the distribution of linear velocities through the membrane in the XT140 capsule is independent of the feed flow rate and the type of protein transmitted. Introduction of this finding into the ZRM permits quantitative predictions of breakthrough over a range of industrially relevant operating conditions. Biotechnol. Bioeng. 2014;111: 1587–1594. © 2014 Wiley Periodicals, Inc.     

A review of thermochemical upgrading of pyrolysis bio‐oil: Techno‐economic analysis,life cycle assessment,and technology readiness

Technologies for upgrading fast pyrolysis bio‐oil to drop‐in fuels and coproducts are under development and show promise for decarbonizing energy supply for transportation and chemicals markets. The successful commercialization of these fuels and the technologies deployed to produce them depend on production costs, scalability, and yield. To meet environmental regulations, pyrolysis‐based biofuels need to adhere to life cycle greenhouse gas intensity standards relative to their petroleum‐based counterparts. We review literature on fast pyrolysis bio‐oil upgrading and explore key metrics that influence their commercial viability through life cycle assessment (LCA) and techno‐economic analysis (TEA) methods together with technology readiness level (TRL) evaluation. We investigate the trade‐offs among economic, environmental, and technological metrics derived from these methods for individual technologies as a means of understanding their nearness to commercialization. Although the technologies reviewed have not attained commercial investment, some have been pilot tested. Predicting the projected performance at scale‐up through models can, with industrial experience, guide decision‐making to competitively meet energy policy goals. LCA and TEA methods that ensure consistent and reproducible models at a given TRL are needed to compare alternative technologies. This study highlights the importance of integrated analysis of multiple economic, environmental, and technological metrics for understanding performance prospects and barriers among early stage technologies.     

Scaling up microbial fuel cells and other bioelectrochemical systems

Scientific research has advanced on different microbial fuel cell (MFC) technologies in the laboratory at an amazing pace, with power densities having reached over 1 kW/m³ (reactor volume) and to 6.9 W/m² (anode area) under optimal conditions. The main challenge is to bring these technologies out of the laboratory and engineer practical systems for bioenergy production at larger scales. Recent advances in new types of electrodes, a better understanding of the impact of membranes and separators on performance of these systems, and results from several new pilot-scale tests are all good indicators that commercialization of the technology could be possible within a few years. Some of the newest advances and future challenges are reviewed here with respect to practical applications of these MFCs for renewable energy production and other applications.     

Sodium‐Ion Battery Materials and Electrochemical Properties Reviewed

The demand for electrochemical energy storage technologies is rapidly increasing due to the proliferation of renewable energy sources and the emerging markets of grid‐scale battery applications. The properties of batteries are ideal for most electrical energy storage (EES) needs, yet, faced with resource constraints, the ability of current lithium‐ion batteries (LIBs) to match this overwhelming demand is uncertain. Sodium‐ion batteries (SIBs) are a novel class of batteries with similar performance characteristics to LIBs. Since they are composed of earth‐abundant elements, cheaper and utility scale battery modules can be assembled. As a result of the learning curve in the LIB technology, a phenomenal progression in material development has been realized in the SIB technology. In this review, innovative strategies used in SIB material development, and the electrochemical properties of anode, cathode, and electrolyte combinations are elucidated. Attractive performance characteristics are herein evidenced, based on comparative gravimetric and volumetric energy densities to state‐of‐the‐art LIBs. In addition, opportunities and challenges toward commercialization are herein discussed based on patent data trend analysis. With extensive industrial adaptations expected, the commercial prospects of SIBs look promising and this once discarded technology is set to play a major role in EES applications.     

Industrial‐strength ecology: trade‐offs and opportunities in algal biofuel production

Microalgae represent one of the most promising groups of candidate organisms for replacing fossil fuels with contemporary primary production as a renewable source of energy. Algae can produce many times more biomass per unit area than terrestrial crop plants, easing the competing demands for land with food crops and native ecosystems. However, several aspects of algal biology present unique challenges to the industrial‐scale aquaculture of photosynthetic microorganisms. These include high susceptibility to invading aquatic consumers and weeds, as well as prodigious requirements for nutrients that may compete with the fertiliser demands of other crops. Most research on algal biofuel technologies approaches these problems from a cellular or genetic perspective, attempting either to engineer or select algal strains with particular traits. However, inherent functional trade‐offs may limit the capacity of genetic selection or synthetic biology to simultaneously optimise multiple functional traits for biofuel productivity and resilience. We argue that a community engineering approach that manages microalgal diversity, species composition and environmental conditions may lead to more robust and productive biofuel ecosystems. We review evidence for trade‐offs, challenges and opportunities in algal biofuel cultivation with a goal of guiding research towards intensifying bioenergy production using established principles of community and ecosystem ecology.     

Light it up: highly efficient multigene delivery in mammalian cells

Multigene delivery and expression systems are emerging as key technologies for many applications in contemporary biology. We have developed new methods for multigene delivery and expression in eukaryotic hosts for a variety of applications, including production of protein complexes for structural biology and drug development, provision of multicomponent protein biologics, and cell-based assays. We implemented tandem recombineering to facilitate rapid generation of multicomponent gene expression constructs for efficient transformation of mammalian cells, resulting in homogenous cell populations. Analysis of multiple parameters in living cells may require co-expression of fluorescently tagged sensors simultaneously in a single cell, at defined and ideally controlled ratios. Our method enables such applications by overcoming currently limiting challenges. Here, we review recent multigene delivery and expression strategies and their exploitation in mammalian cells. We discuss applications in drug discovery assays, interaction studies, and biologics production, which may benefit in the future from our novel approach.     

Advances and challenges in the production of extracellular thermoduric pullulanases by wild-type and recombinant microorganisms: a review

Thermoduric pullulanases, acting as starch-debranching enzymes, are required in many industrial applications, mainly in the production of concentrated glucose, maltose, and fructose syrups. To date, however, a single pullulanase, from Bacillus acidopullulyticus, is available on the market for industrial purposes. This review is an investigation of the major advances as well as the major challenges being faced with regard to optimization of the production of extracellular thermoduric pullulanases either by their original hosts or by recombinant organisms. The critical aspects linked to industrial pullulanase production, which should always be considered, are emphasized, including those parameters influencing solubility, thermostability, and catalytic efficiency of the enzyme. This review provides new insights for improving the production of extracellular thermoduric pullulanases in the hope that such information may facilitate their commercial utilization and potentially be applied to the development of other industrially relevant enzymes.     

High impact biowastes from South European agro-industries as feedstock for second-generation biorefineries

Availability of bio-based chemicals, materials and energy at reasonable cost will be one of the forthcoming issues for the EU economy. In particular, the development of technologies making use of alternative resources to fossil fuels is encouraged by the current European research and innovation strategy to face the societal challenge of natural resource scarcity, fossil resource dependence and sustainable economic growth. In this respect, second- generation biorefineries, i.e. biorefineries fed with biowastes, appear to be good candidates to substitute and replace the present downstream processing scheme. Contrary to first-generation biorefineries, which make use of dedicated crops or primary cultivations to achieve such a goal, the former employ agricultural, industrial, zootechnical, fishery and forestry biowastes as the main feedstock. This leaves aside any ethical and social issue generated by first-generation approaches, and concomitantly prevents environmental and economical issues associated with the disposal of the aforementioned leftovers. Unfortunately, to date, a comprehensive and updated mapping of the availability and potential use of bioresources for second-generation biorefineries in Europe is missing. This is a lack that severely limits R&D and industrial applications in the sector. On the other hand, attempts at valorizing the most diverse biowastes dates back to the nineteenth century and plenty of information in the literature on their sustainable exploitation is available. However, the large majority of these investigations have been focused on single fractions of biowastes or single steps of biowaste processing, preventing considerations on an integrated and modular (cascade) approach for the whole valorization of organic leftovers. This review aims at addressing these issues by gathering recent data on (a) some of the main high-impact biowastes located in Europe and in particular in its Southern part, and (b) the bio-based chemicals, materials and fuels that can be produced from such residues. In particular, we focused on those key compounds referred to as “chemical platforms”, which have been indicated as fundamental to generate the large majority of the industrially relevant goods to date.