Production Of High-value Compounds Research Articles

Comparative Genomic and Transcriptomic Analyses of Microalgae as Biofactories

Microalgae are unicellular photo- and heterotrophic organisms that, being efficient producers of a rich and complex biomass, have attracted global interest as biofactories for the production of high-value compounds (e.g. PUFAs, proteins and pigments) that can find application in nutraceutical, pharmaceutical and food industries. Microalgae have also been extensively studied in the last two decades, as potential producers of biofuels. However, based on current technologies, this production process is not economically sustainable yet. In this frame, the recent advances in genomic and transcriptomic characterization of microalgae can represent the key to understand the metabolic mechanisms underlying the growth regulation and the biosynthesis of those valuable metabolites, in order to improve the use of microalgae as biofactories.

Production of Oil in Plant Vegetative Tissues

Synthetic biology is an emerging area of science focused on the development of well‐defined molecular components that can be assembled and utilized for rational engineering design. In many cases, the process is geared towards production of high‐value compounds, and in biofuels research, a major goal is to maximize the amount of oil that can be recovered from plants. While seeds are the traditional source of plant oil, we and others are considering the engineering of oil in plant vegetative tissues. The rationale is that the biomass of plants is dominated by leaves and stems, and as such, production of even modest amounts of oil in these tissues may significantly increase the amount of oil recovered from plants. In this seminar I will describe a variety of approaches that we are using to increase the steady‐state amount of oil in plant leaves, including enhancement of the mechanisms for oil synthesis and disruption of processes for oil breakdown. Furthermore, we have recently identified proteins involved in lipid droplet biogenesis and compartmentation, which provide additional tools for modulating the packaging and storage of lipids in plant cells. Collectively, these studies define a growing set of molecular components that can be used for lipid modification. The results of these studies are informing not only the engineering of oil content in plant leaves, but also providing new insights to oil production in plant seeds. Implications for both food and fuel will be discussed.

Establishing Chlamydomonas reinhardtii as an industrial biotechnology host

Microalgae constitute a diverse group of eukaryotic unicellular organisms that are of interest for pure and applied research. Owing to their natural synthesis of value-added natural products microalgae are emerging as a source of sustainable chemical compounds, proteins and metabolites, including but not limited to those that could replace compounds currently made from fossil fuels. For the model microalga, Chlamydomonas reinhardtii, this has prompted a period of rapid development so that this organism is poised for exploitation as an industrial biotechnology platform. The question now is how best to achieve this? Highly advanced industrial biotechnology systems using bacteria and yeasts were established in a classical metabolic engineering manner over several decades. However, the advent of advanced molecular tools and the rise of synthetic biology provide an opportunity to expedite the development of C. reinhardtii as an industrial biotechnology platform, avoiding the process of incremental improvement. In this review we describe the current status of genetic manipulation of C. reinhardtii for metabolic engineering. We then introduce several concepts that underpin synthetic biology, and show how generic parts are identified and used in a standard manner to achieve predictable outputs. Based on this we suggest that the development of C. reinhardtii as an industrial biotechnology platform can be achieved more efficiently through adoption of a synthetic biology approach.Significance StatementChlamydomonas reinhardtii offers potential as a host for the production of high value compounds for industrial biotechnology. Synthetic biology provides a mechanism to generate generic, well characterised tools for application in the rational genetic manipulation of organisms: if synthetic biology principles were adopted for manipulation of C. reinhardtii, development of this microalga as an industrial biotechnology platform would be expedited.

Highly efficient, long life, reusable and robust photosynthetic hybrid core–shell beads for the sustainable production of high value compounds

An efficient one-step process to synthesize highly porous (Ca-alginate–SiO2–polycation) shell: (Na-alginate–SiO2) core hybrid beads for cell encapsulation, yielding a reusable long-life photosynthetically active material for a sustainable manufacture of high-value metabolites is presented. Bead formation is based on crosslinking of an alginate biopolymer and mineralisation of silicic acid in combination with a coacervation process between a polycation and the silica sol, forming a semi-permeable external membrane. The excellent mechanical strength and durability of the monodispersed beads and the control of their porosity and textural properties is achieved by tailoring the silica and alginate loading, polycation concentration and incubation time during coacervation. This process has led to the formation of a remarkably robust hybrid material that confers exceptional protection to live cells against sheer stresses and contamination in a diverse range of applications. Dunaliella tertiolecta encapsulated within this hybrid core–shell system display high photosynthetic activity over a long duration (>1year). This sustainable biotechnology could find use in high value chemical harvests and biofuel cells to photosynthetic solar cells (energy transformation, electricity production, water splitting technologies). Furthermore the material can be engineered into various forms from spheres to variable thickness films, broadening its potential applications.

Transcriptomic and proteomic dynamics in the metabolism of a diazotrophic cyanobacterium, Cyanothece sp. PCC 7822 during a diurnal light-dark cycle.

BackgroundCyanothece sp. PCC 7822 is an excellent cyanobacterial model organism with great potential to be applied as a biocatalyst for the production of high value compounds. Like other unicellular diazotrophic cyanobacterial species, it has a tightly regulated metabolism synchronized to the light–dark cycle. Utilizing transcriptomic and proteomic methods, we quantified the relationships between transcription and translation underlying central and secondary metabolism in response to nitrogen free, 12 hour light and 12 hour dark conditions.ResultsBy combining mass-spectrometry based proteomics and RNA-sequencing transcriptomics, we quantitatively measured a total of 6766 mRNAs and 1322 proteins at four time points across a 24 hour light–dark cycle. Photosynthesis, nitrogen fixation, and carbon storage relevant genes were expressed during the preceding light or dark period, concurrent with measured nitrogenase activity in the late light period. We describe many instances of disparity in peak mRNA and protein abundances, and strong correlation of light dependent expression of both antisense and CRISPR-related gene expression. The proteins for nitrogenase and the pentose phosphate pathway were highest in the dark, whereas those for glycolysis and the TCA cycle were more prominent in the light. Interestingly, one copy of the psbA gene encoding the photosystem II (PSII) reaction center protein D1 (psbA4) was highly upregulated only in the dark. This protein likely cannot catalyze O2 evolution and so may be used by the cell to keep PSII intact during N2 fixation. The CRISPR elements were found exclusively at the ends of the large plasmid and we speculate that their presence is crucial to the maintenance of this plasmid.ConclusionsThis investigation of parallel transcriptional and translational activity within Cyanothece sp. PCC 7822 provided quantitative information on expression levels of metabolic pathways relevant to engineering efforts. The identification of expression patterns for both mRNA and protein affords a basis for improving biofuel production in this strain and for further genetic manipulations. Expression analysis of the genes encoded on the 6 plasmids provided insight into the possible acquisition and maintenance of some of these extra-chromosomal elements.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1185) contains supplementary material, which is available to authorized users.

Green and sustainable production of high value compounds via a microalgae encapsulation technology that relies on CO2as a principle reactant

A very promising and facile one-pot synthesis pathway is presented for the microencapsulation of live cells in a very porous core–shell system based upon a robust matrix. (Alginate–SiO2–polycation) shell@(alginate–SiO2) core hybrid beads, on the millimeter scale, containing live cells are obtained through cross-linking chemistry and the polycondensation of silicic acid in conjunction with the use of a polycation to negate the surface charge on silica. Very interestingly it is revealed that the polycation used (PDADMAC) plays a very important role in the formation of highly robust core–shell beads. PDADMAC acts as a catalyst in the polycondensation of silicic acid, leading to the formation of a resistant double layer shell comprising of an interior layer of alginate–SiO2 with a very homogeneous distribution of porous SiO2 and an external layer of porous PDADMAC that confines SiO2 within the bead. The photosynthetic chlorophyta Dunaliella tertiolecta, which produces high value metabolites (such as anti-oxidants, pharmacologically active compounds, neutraceuticals etc.) via photosynthesis, has been encapsulated within this core–shell system. Oximetry and fluorescence measurements highlight how this algal culture can remain photosynthetically active over an extraordinarily long period of 13 months for high value compound production, whilst entrapped within a highly porous, mechanically and chemically stable, optically transparent matrix, with no observable leaching of the cells from the core of the beads. HPLC has been employed to highlight the presence of excreted metabolites, based on neutral sugar building blocks such as arabinose, galactose and xylose, in the surrounding media. These results reveal how this kind of high performance, low-cost, and easily scaleable core–shell living material could be employed in large scale photobioreactors (PBRs), to potentially facilitate metabolite harvesting whilst protecting the culture from external contamination and for green energy production and environmental (CO2) remediation.

Observer-Based Backstepping Controller for Microalgae Cultivation

Microalgae are microscopic plants that exist in an aquatic environment. They are involved in the production of high value compounds and also have applications in energy production. In this work, the regulation of biomass concentration in a bioreactor is investigated by using an observer-based backstepping control approach. The process is controlled to operate in a constant biomass concentration mode, in order to maintain the culture at a desired concentration and to sustain high biomass production levels. Combined with the backstepping controller, a nonlinear Lipschitz observer is proposed. On the basis of the Droop model, which describes the dynamic behavior of the microalgae process and based on the biomass measurements, the stability of the state error dynamics can be guaranteed. Finally, simulations and comparisons with a PI controller are developed to show the performance of the nonlinear observer-based controller in set point tracking and load rejection in the presence of parameter uncertainties.

Optimizing microalgal production in raceway systems

The industrial exploitation of microalgae is characterized by the production of high-value compounds. Optimization of the performance of microalgae culture systems is essential to render the process economically viable. For raceway systems, the optimization based on optimal control theory is rather challenging, because the process is by essence periodically forced and, as a consequence, optimization must be carried out in a periodic framework. In this article, we propose a simple operational criterion for raceway systems that when integrated in a strategy of closed-loop control allows attaining biomass productivities very near to the theoretical maximal productivities. The strategy developed was tested numerically using a mathematical model of microalgae growth in raceways. The model takes into account the temporal variation of the environmental variables temperature and light intensity and their influence on microalgae growth.

Output Feedback Passivity-Based Controller for Microalgae Cultivation

Microalgae are microscopic plants existing in aquatic environment. They can be used in the production of high value compounds and they have promising opportunities in energy production, wastewater treatment and fixation of carbon dioxide. In this context, the control of the cultivation requires a special attention in order to obtain high amounts of biomass. The control of microalgae cultivation is approached from passivity-based control perspectives. The proposed controller solves set point tracking and stabilizes the control loop by the passivity properties. Moreover, in order to obtain the process state variables from the measurement output, which are used in the control law, a nonlinear observer with guaranteed stability of estimation error dynamics is proposed. The design of the nonlinear controller and the observer is based on the Droop model, describing the dynamic behaviour of the microalgae process in a practical way. Finally, the performance of the nonlinear observer-based controller is shown by simulation.

Increased production of azadirachtin from an improved method of androgenic cultures of a medicinal tree Azadirachta indica A. Juss.

This report is the first direct evidence of azadirachtin production in androgenic haploid cultures of Azadirachta indica, a woody medicinal tree. Anther cultures at early-late-uninucleate stage of microspores were established on MS medium with BAP (5 µM), 2,4-D (1 µM) and NAA (1 µM) containing 12% sucrose. The calli, induced, were further multiplied on 2,4-D and Kinetin media. Shoots, differentiated on BAP (2.2 µM) + NAA (0.05 µM) medium, were elongated on MS + BAP (0.5 µM) and multiplied on MS + BAP (1 µM) + CH (250 mg/l). Thereafter, the shoots were rooted on 1/4 MS + IBA (0.5 µM). Cytological analysis of the calli and regenerants have confirmed their haploid status with the chromosome number as 2n=x=12. The haploid cell lines and leaves from in vitro grown plantlets were analyzed for azadirachtin by RP-HPLC and mass spectroscopy. Maximum azadirachtin (728.41 µg/g DW) was detected in calli supporting best shoot proliferation while least (49 µg/g DW) was observed in an undifferentiated line from maintenance medium. This study has brought us a step closer to develop genetically pure lines that could serve as new and attractive alternative ways of homogeneous controlled production of high value compounds, round the year, independent of geographical and climatic barrier.

Growth and solvent production by Clostridium pasteurianum ATCC® 6013™ utilizing biodiesel‐derived crude glycerol as the sole carbon source

AbstractThe rapidly expanding market for biodiesel is dramatically altering the cost and availability of glycerol, as typical biodiesel production processes generate about 10 wt % glycerol. Biodiesel producers have little financial incentive to purify the crude glycerol, and because it contains methanol, crude glycerol is considered a hazardous waste. Therefore, it is critical that new and innovative processes for managing and utilizing the glycerol co‐product be developed. In the longer term, as supplies continue to increase, glycerol will become a versatile building block chemical for the production of high value compounds within an integrated biorefinery.This research investigated the value‐added conversion of crude glycerol generated during biodiesel production using anaerobic fermentation. Pure cultures of Clostridium pasteurianum have been shown to produce significant amounts of butanol and 1,3‐propanediol when utilizing purified glycerol as the sole substrate. Butanol is of particular interest as a renewable biofuel, as it has a higher heating value, higher octane number, lower vapor pressure, and higher miscibility than ethanol, making butanol preferable to ethanol for blending with petroleum fuels.Preliminary experiments compared growth and product formation of C. pasteurianum (ATCC®6013™) utilizing both purified and biodiesel‐derived crude glycerol as the sole carbon source in batch culture. Cultures utilizing crude glycerol demonstrated significant growth and production of butanol, 1,3‐PDO, and ethanol at concentrations up to 25 g/L crude glycerol. This work is significant in that it is the first report of butanol production from crude glycerol using this organism. The maximum yield of butanol produced from pure glycerol was 0.36 g/g, while the maximum butanol yield produced from crude glycerol was 0.30 g/g. These yields are substantially higher than the 0.15–0.20 g/g butanol yield typically achieved during the fermentation of glucose using C. acetobutylicum. These results indicate that biodiesel‐derived crude glycerol is a promising, low‐cost, renewable feedstock for butanol production. © 2009 American Institute of Chemical Engineers Environ Prog, 2009

Unscented Kalman Filter state and parameter estimation in a photobioreactor for microalgae production

Microalgae have many applications such as the production of high value compounds (source of long-chain polyunsaturated fatty acids, vitamins, and pigments), in energy production (e.g. photobiological hydrogen, biofuel, methane) or in environmental remediation (especially carbon dioxide fixation and greenhouse gas emissions reduction). However, the photobioreactor microalgae process needs complex and costly hardware sensors, especially for biomass measurement. Thus, state and parameter estimation seems to be a critical issue and is studied in this paper in the case of a culture of the microalga Porphyridium purpureum. This paper is an extension of the previous work of Becerra-Celis et al. (2008) where the principal objective is to design a biomass estimator of this microalga production in a photobioreactor based on the total inorganic carbon measurement. Unscented Kalman filtering is applied to estimation of states and model parameters, producing better performances in comparison with Extented Kalman filtering. Numerical simulations in batch mode, and real-life experiments in continuous mode have been carried out. Corresponding results are given in order to highlight the performance of the proposed estimator.

Shortening the Light Path Length of Photobioreactors to Elevate the Cell Mass Concentration of Cyanobacterium Synechococcus sp. Strain PCC6301

To explore high-cell density cultivation of photolithotrophs in the production of high-value compounds, a model cyanobacterium Synechococcus sp. strain PCC6301 was grown in rectangular photobioreactors with light path lengths of 0.1, 0.3, 0.5, 1.4 and 5 cm. A mathematical model to correlate cell growth with medium concentration, light transmittance and light path length was formulated. Logarithmic and linear growth phase were prolonged, and linear growth rate was elevated by shortening the light path length and by avoiding monohydrogen phosphate ion depletion. With sufficient monohydrogen phosphate ions, a high cell mass concentration of 24.5 mg·mL–1 was accomplished using a 0.1 cm light path length photobioreactor.

Estimation of microalgal photobioreactor production based on total inorganic carbon in the medium

Microalgae biotechnology has been focusing on the use of algae in the production of high value compounds. During the last few decades, the intense research effort has been aiming at improving new controls and supervising tools as well as on a good process understanding. This requirement involves a large diversity and a better accessibility to process measurements. Probes or sensors are required to control the process. They are however relatively limited. Classical photobioreactors are usually equipped with temperature, dissolved oxygen and pH probes. These sensors actually provide very little online information on cell growth, viability, metabolic state and production. For the time being, sensors reliability cannot meet industrial bioprocessing requirements. In this context software sensors show numerous potentialities. The central axis of this work is the development of an extended Kalman filter (EKF) for the estimation of biomass concentration based on a dynamic process model in combination with total inorganic carbon measurement. A microalga Porphyridium purpureum was used as a model organism in this study. Numerical simulations and real-life experiments (batch and continuous mode) have been carried out and corresponding results are given in order to highlight the performance of the proposed estimator.

Directed microspore‐specific recombination of transgenic alleles to prevent pollen‐mediated transmission of transgenes

A major challenge for future genetically modified (GM) crops is to prevent undesired gene flow of transgenes to plant material intended for another use. Recombinase-mediated auto excision of transgenes directed by a tightly controlled microspore-specific promoter allows efficient removal of either the selectable marker gene or of all introduced transgenes during microsporogenesis. This way, transgene removal becomes an integral part of the biology of pollen maturation, not requiring any external stimulus such as chemical induction by spraying. We here show the feasibility of engineering transgenic plants to produce pollen devoid of any transgene. Highly efficient excision of transgenes from tobacco pollen was achieved with a potential failure rate of at most two out of 16,800 seeds (0.024%). No evidence for either premature activation or absence of activation of the recombinase system was observed under stress conditions in the laboratory. This approach can prevent adventitious presence of transgenes in non-GM crops or related wild species by gene flow. Such biological containment may help the deployment and management of coexistence practices to support consumer choice and will promote clean molecular farming for the production of high-value compounds in plants.

Production of high-value compounds: carotenoids and vitamin E

The unraveling in recent years of the pathways and genes that are responsible for biosynthesis of vitamins and carotenoids in plants has provided new molecular tools that can be utilized to genetically modify the content and composition of these compounds in vivo. There have been recent examples of successful manipulation of carotenoid composition and vitamin E activity in plants using gene-transfer technology.