Engineered ketocarotenoid biosynthesis in the polyextremophilic red microalga Cyanidioschyzon merolae 10D.

The polyextremophilic Cyanidiophyceae are eukaryotic red microalgae with promising biotechnological properties arising from their low pH and elevated temperature requirements which can minimize culture contamination at scale. Cyanidioschyzon merolae 10D is a cell wall deficient species with a fully sequenced genome that is amenable to nuclear transgene integration by targeted homologous recombination. C. merolae maintains a minimal carotenoid profile and here, we sought to determine its capacity for ketocarotenoid accumulation mediated by heterologous expression of a green algal ß-carotene ketolase (BKT) and hydroxylase (CHYB). To achieve this, a synthetic transgene expression cassette system was built to integrate and express Chlamydomonas reinhardtii (Cr) sourced enzymes by fusing native C. merolae transcription, translation and chloroplast targeting signals to codon-optimized coding sequences. Chloramphenicol resistance was used to select for the integration of synthetic linear DNAs into a neutral site within the host genome. CrBKT expression caused accumulation of canthaxanthin and adonirubin as major carotenoids while co-expression of CrBKT with CrCHYB generated astaxanthin as the major carotenoid in C. merolae. Unlike green algae and plants, ketocarotenoid accumulation in C. merolae did not reduce total carotenoid contents, but chlorophyll a reduction was observed. Light intensity affected global ratios of all pigments but not individual pigment compositions and phycocyanin contents were not markedly different between parental strain and transformants. Continuous illumination was found to encourage biomass accumulation and all strains could be cultivated in simulated summer conditions from two different extreme desert environments. Our findings present the first example of carotenoid metabolic engineering in a red eukaryotic microalga and open the possibility for use of C. merolae 10D for simultaneous production of phycocyanin and ketocarotenoid pigments.

Assessing Subsoil Conditions with an ASABE Conform Vertical Penetrometer-Development and Evaluation.

Soil is the habitat for soil organisms and associated soil physical and chemical processes. The subsoil is a large reserve of water and nutrients. Soil and subsoil are thus significantly involved in the yield capacity of a site and its resilience in the case of unfavorable weather conditions. Subsoil can also retain water in drought phases and stores carbon. In times of climate change and scarcity of resources, many scientific activities involve subsoil and require sensors to assess subsoil conditions and properties. An electrically driven penetrometer with an integrated soil water content sensor could be an appropriate tool for such applications; however, such a subsoil measurement tool does not exist. One major reason for this is that, when penetrating compacted subsoil, high penetration forces (including friction) act on the penetrating thin rod (diameter 1 cm). The development of a tractor-mounted subsoil penetrometer for depths up to 2 m is described in this study. An ASABE standard cone is implemented, which can access heavy compacted layers. The rod, which includes wires for embedding an FDI moisture sensor in the cone tip, is covered by a protection tube. The penetration resistance measurement can be performed without being influenced by shaft friction. The rod, along with the sensor, is implemented in a tower that can be shifted laterally and can take probes in a single line without moving the tractor. To confirm the quality of the developed subsoil penetrometer, a suitable evaluation method is presented. Typical arable soil (loamy silt) was filled in boxes and compacted homogeneously using a hydraulic stamp so that different setups of the penetrometer could be compared and evaluated. The evaluation concludes that the distance between the free cone tip and the protection shaft should be at least 10 cm to measure the penetration resistance of soil without being influenced by the protection tube. Furthermore, the developed penetrometer has sufficient stability and precision for accessing subsoil. In field trials, the subsoil penetrometer was compared with a standard penetrometer and has proved its suitability.

Capability of the TrueColor Sensor Array for Determining the Nitrogen Supply in Winter Barley (Hordeum vulgare L.).

In agriculture, efforts are being made to reduce pesticides and fertilizers because of the possible negative environmental impacts, high costs, political requirements, and declining social acceptance. With precision farming, significant savings can be achieved by the site-specific application of fertilizers. In contrast to currently available single sensors and camera-based systems, arrays or line sensors provide a suitable spatial resolution without requiring complex signal processing and promise significant potential regarding price and precision. Such systems comprise a cost-effective and compact unit that can be extended to any working width by cascading into arrays. In this study, experiments were performed to evaluate the applicability of a TrueColor sensor array in monitoring the nitrogen supply of winter barley during its growth. This sensor is based on recording the reflectance values in various channels of the CIELab color space: luminosity, green-red, and blue-yellow. The unique selling point of this sensor is the detection of luminosity because only the CIELab color space provides this opportunity. Strong correlations were found between the different reflection channels and the nitrogen level (R² = 0.959), plant coverage (R² = 0.907), and fresh mass yield (R² = 0.866). The fast signal processing allows this sensor to meet stringent demands for the operating speed, spatial resolution, and price structure.

The genomes of polyextremophilic cyanidiales contain 1% horizontally transferred genes with diverse adaptive functions.

The role and extent of horizontal gene transfer (HGT) in eukaryotes are hotly disputed topics that impact our understanding of the origin of metabolic processes and the role of organelles in cellular evolution. We addressed this issue by analyzing 10 novel Cyanidiales genomes and determined that 1% of their gene inventory is HGT-derived. Numerous HGT candidates share a close phylogenetic relationship with prokaryotes that live in similar habitats as the Cyanidiales and encode functions related to polyextremophily. HGT candidates differ from native genes in GC-content, number of splice sites, and gene expression. HGT candidates are more prone to loss, which may explain the absence of a eukaryotic pan-genome. Therefore, the lack of a pan-genome and cumulative effects fail to provide substantive arguments against our hypothesis of recurring HGT followed by differential loss in eukaryotes. The maintenance of 1% HGTs, even under selection for genome reduction, underlines the importance of non-endosymbiosis related foreign gene acquisition.

A True-Color Sensor and Suitable Evaluation Algorithm for Plant Recognition.

Plant-specific herbicide application requires sensor systems for plant recognition and differentiation. A literature review reveals a lack of sensor systems capable of recognizing small weeds in early stages of development (in the two- or four-leaf stage) and crop plants, of making spraying decisions in real time and, in addition, are that are inexpensive and ready for practical use in sprayers. The system described in this work is based on free cascadable and programmable true-color sensors for real-time recognition and identification of individual weed and crop plants. The application of this type of sensor is suitable for municipal areas and farmland with and without crops to perform the site-specific application of herbicides. Initially, databases with reflection properties of plants, natural and artificial backgrounds were created. Crop and weed plants should be recognized by the use of mathematical algorithms and decision models based on these data. They include the characteristic color spectrum, as well as the reflectance characteristics of unvegetated areas and areas with organic material. The CIE-Lab color-space was chosen for color matching because it contains information not only about coloration (a- and b-channel), but also about luminance (L-channel), thus increasing accuracy. Four different decision making algorithms based on different parameters are explained: (i) color similarity (ΔE); (ii) color similarity split in ΔL, Δa and Δb; (iii) a virtual channel 'd' and (iv) statistical distribution of the differences of reflection backgrounds and plants. Afterwards, the detection success of the recognition system is described. Furthermore, the minimum weed/plant coverage of the measuring spot was calculated by a mathematical model. Plants with a size of 1-5% of the spot can be recognized, and weeds in the two-leaf stage can be identified with a measuring spot size of 5 cm. By choosing a decision model previously, the detection quality can be increased. Depending on the characteristics of the background, different models are suitable. Finally, the results of field trials on municipal areas (with models of plants), winter wheat fields (with artificial plants) and grassland (with dock) are shown. In each experimental variant, objects and weeds could be recognized.

Co-liquefaction of mixed culture microalgal strains under sub-critical water conditions.

We report the co-liquefaction performance of unicellular, red alga Cyanidioschyzon merolae and Galdieria sulphuraria under sub-critical water conditions within a stainless-steel batch reactor under different temperatures (150-300°C), residence time (15-60min), and Cyanidioschyzon merolae to Galdieria sulphuraria mass loading (0-100%). Individual liquefaction of C. merolae and G. sulphuraria at 300°C achieved maximum biocrude oil yield of 18.9 and 14.0%, respectively. The yield of biocrude oil increased to 25.5%, suggesting a positive synergistic effect during the co-liquefaction of 80-20mass loading of C. merolae to G. sulphuraria. The biocrude oils were analyzed by FT-ICR MS which showed that co-liquefaction did not significantly affect the distribution of product compounds compared to individual oils. The co-liquefied biocrude and biochar have a higher-heating-value of 35.28 and 7.96MJ/kg. Ultimate and proximate analysis were performed on algae biomass, biocrude and biochar.

Hydrothermal liquefaction of Cyanidioschyzon merolae and the influence of catalysts on products.

This work investigates the hydrothermal liquefaction (HTL) of Cyanidioschyzon merolae algal species under various reaction temperatures and catalysts. Liquefaction of microalgae was performed with 10% solid loading for 30min at temperatures of 180-300°C to study the influences of two base and two acid catalysts on HTL product fractions. Maximum biocrude oil yield of 16.98% was obtained at 300°C with no catalyst. The biocrude oil yield increased to 22.67% when KOH was introduced into the reaction mixture as a catalyst. The algal biocrude and biochar has a higher heating values (HHV) of 32.22MJkg-1 and 20.78MJkg-1 respectively when no catalyst was used. Gas chromatography time of flight mass spectrometry (GC/TOFMS) was employed to analyze the biocrude oil composition, and elemental analysis was performed on the algae, biocrude and biochar samples. Analysis of the HTL aqueous phase revealed the presence of valuable products.

An Assessment of Three Different In Situ Oxygen Sensors for Monitoring Silage Production and Storage.

Oxygen (O2) concentration inside the substrate is an important measurement for silage-research and-practical management. In the laboratory gas chromatography is commonly employed for O2 measurement. Among sensor-based techniques, accurate and reliable in situ measurement is rare because of high levels of carbon dioxide (CO2) generated by the introduction of O2 in the silage. The presented study focused on assessing three types of commercial O2 sensors, including Clark oxygen electrodes (COE), galvanic oxygen cell (GOC) sensors and the Dräger chip measurement system (DCMS). Laboratory cross calibration of O2 versus CO2 (each 0-15 vol.%) was made for the COE and the GOC sensors. All calibration results verified that O2 measurements for both sensors were insensitive to CO2. For the O2 in situ measurement in silage, all O2 sensors were first tested in two sealed barrels (diameter 35.7 cm; height: 60 cm) to monitor the O2 depletion with respect to the ensiling process (Test-A). The second test (Test-B) simulated the silage unloading process by recording the O2 penetration dynamics in three additional barrels, two covered by dry ice (0.6 kg or 1.2 kg of each) on the top surface and one without. Based on a general comparison of the experimental data, we conclude that each of these in situ sensor monitoring techniques for O2 concentration in silage exhibit individual advantages and limitations.

Closed-Loop Control of Chemical Injection Rate for a Direct Nozzle Injection System.

To realize site-specific and variable-rate application of agricultural pesticides, accurately metering and controlling the chemical injection rate is necessary. This study presents a prototype of a direct nozzle injection system (DNIS) by which chemical concentration transport lag was greatly reduced. In this system, a rapid-reacting solenoid valve (RRV) was utilized for injecting chemicals, driven by a pulse-width modulation (PWM) signal at 100 Hz, so with varying pulse width the chemical injection rate could be adjusted. Meanwhile, a closed-loop control strategy, proportional-integral-derivative (PID) method, was applied for metering and stabilizing the chemical injection rate. In order to measure chemical flow rates and input them into the controller as a feedback in real-time, a thermodynamic flowmeter that was independent of chemical viscosity was used. Laboratory tests were conducted to assess the performance of DNIS and PID control strategy. Due to the nonlinear input-output characteristics of the RRV, a two-phase PID control process obtained better effects as compared with single PID control strategy. Test results also indicated that the set-point chemical flow rate could be achieved within less than 4 s, and the output stability was improved compared to the case without control strategy.

Algal-based, single-step treatment of urban wastewaters.

Currently, urban wastewaters (UWW) laden with organic carbon (BOD) and nutrients (ammoniacal nitrogen, N, and phosphates, P) are treated in multi-stage, energy-intensive process trains to meet the mandated discharge standards. This study presents a single-step process based on mixotrophic metabolism for simultaneous removal of carbon and nutrients from UWWs. The proposed system is designed specifically for hot, arid environments utilizing an acidophilic, thermotolerant algal species, Galdieria sulphuraria, and an enclosed photobioreactor to limit evaporation. Removal rates of BOD, N, and P recorded in this study (14.93, 7.23, and 1.38 mg L(-1) d(-1), respectively) are comparable to literature reports. These results confirm that the mixotrophic system can reduce the energy costs associated with oxygen supply in current UWW treatment systems, and has the potential to generate more energy-rich biomass for net energy extraction from UWW.

An improved sensor for precision detection of in situ stem water content using a frequency domain fringing capacitor.

One role of stems is that of water storage. The water content of stems increases and decreases as xylem water potential increases and decreases, respectively. Hence, a nondestructive method to measure stem water content (StWC) = (volume of water) : (volume of stem), could be useful in monitoring the drought stress status of plants. We introduce a frequency domain inner fringing capacitor-sensor for measuring StWC which operates at 100 MHz frequency. The capacitor-sensor consists of two wave guides (5-mm-wide braided metal) that snugly fit around the surface of a stem with a spacing of 4-5 mm between guides. Laboratory measurements on analog stems reveals that the DC signal output responds linearly to the relative dielectric constant of the analog stem, is most sensitive to water content between the waveguides to a depth of c. 3 mm from the stem surface, and calibrations based on the gravimetric water loss of excised stems of plants revealed a resolution in StWC of < ± 0.001 v/ v. The sensor performed very well on whole plants with a 100-fold increased resolution compared with previous frequency domain and time domain reflectometry methods and, hence, may be very useful for future research requiring nondestructive measurements of whole plants.

Fast and solvent-free quantitation of boar taint odorants in pig fat by stable isotope dilution analysis-dynamic headspace-thermal desorption-gas chromatography/time-of-flight mass spectrometry.

Boar taint is a specific off-odour of boar meat products, known to be caused by at least three unpleasant odorants, with very low odour thresholds. Androstenone is a boar pheromone produced in the testes, whereas skatole and indole originate from the microbial breakdown of tryptophan in the intestinal tract. A new procedure, applying stable isotope dilution analysis (SIDA) and dynamic headspace-thermal desorption-gas chromatography/time-of-flight mass spectrometry (dynHS-TD-GC/TOFMS) for the simultaneous quantitation of these boar taint compounds in pig fat was elaborated and validated in this paper. The new method is characterised by a simple and solvent-free dynamic headspace sampling. The deuterated compounds d3-androstenone, d3-skatole and d6-indole were used as internal standards to eliminate matrix effects. The method validation performed revealed low limits of detection (LOD) and quantitation (LOQ) with high accuracy and precision, thus confirming the feasibility of the new dynHS-TD-GC/TOFMS approach for routine analysis.

Genome of an arbuscular mycorrhizal fungus provides insight into the oldest plant symbiosis.

The mutualistic symbiosis involving Glomeromycota, a distinctive phylum of early diverging Fungi, is widely hypothesized to have promoted the evolution of land plants during the middle Paleozoic. These arbuscular mycorrhizal fungi (AMF) perform vital functions in the phosphorus cycle that are fundamental to sustainable crop plant productivity. The unusual biological features of AMF have long fascinated evolutionary biologists. The coenocytic hyphae host a community of hundreds of nuclei and reproduce clonally through large multinucleated spores. It has been suggested that the AMF maintain a stable assemblage of several different genomes during the life cycle, but this genomic organization has been questioned. Here we introduce the 153-Mb haploid genome of Rhizophagus irregularis and its repertoire of 28,232 genes. The observed low level of genome polymorphism (0.43 SNP per kb) is not consistent with the occurrence of multiple, highly diverged genomes. The expansion of mating-related genes suggests the existence of cryptic sex-related processes. A comparison of gene categories confirms that R. irregularis is close to the Mucoromycotina. The AMF obligate biotrophy is not explained by genome erosion or any related loss of metabolic complexity in central metabolism, but is marked by a lack of genes encoding plant cell wall-degrading enzymes and of genes involved in toxin and thiamine synthesis. A battery of mycorrhiza-induced secreted proteins is expressed in symbiotic tissues. The present comprehensive repertoire of R. irregularis genes provides a basis for future research on symbiosis-related mechanisms in Glomeromycota.

Comprehensive theory of the Deans' switch as a variable flow splitter: fluid mechanics, mass balance, and system behavior.

The Deans' switch is an effluent switching device based on controlling flows of carrier gas instead of mechanical valves in the analytical flow path. This technique offers high inertness and a wear-free operation. Recently new monolithic microfluidic devices have become available. In these devices the whole flow system is integrated into a small metal device with low thermal mass and leak-tight connections. In contrast to a mechanical valve-based system, a flow-controlled system is more difficult to calculate. Usually the Deans' switch is used to switch one inlet to one of two outlets, by means of two auxiliary flows. However, the Deans' switch can also be used to deliver the GC effluent with a specific split ratio to both outlets. The calculation of the split ratio of the inlet flow to the two outlets is challenging because of the asymmetries of the flow resistances. This is especially the case, if one of the outlets is a vacuum device, such as a mass spectrometer, and the other an atmospheric detector, e.g. a flame ionization detector (FID) or an olfactory (sniffing) port. The capillary flows in gas chromatography are calculated with the Hagen-Poiseuille equation of the laminar, isothermal and compressible flow in circular tubes. The flow resistances in the new microfluidic devices have to be calculated with the corresponding equation for rectangular cross-section microchannels. The Hagen-Poiseuille equation underestimates the flow to a vacuum outlet. A corrected equation originating from the theory of rarefied flows is presented. The calculation of pressures and flows of a Deans' switch based chromatographic system is done by the solution of mass balances. A specific challenge is the consideration of the antidiffusion resistor between the two auxiliary gas lines of the Deans' switch. A full solution for the calculation of the Deans' switch including this restrictor is presented. Results from validation measurements are in good accordance with the developed theories. A spreadsheet-based flow calculator is part of the Supporting Information.

In situ ethyl ester production from wet algal biomass under microwave-mediated supercritical ethanol conditions.

An in situ transesterification approach was demonstrated for converting lipid-rich wet algae (Nannochloropsis salina) into fatty acid ethyl esters (FAEE) under microwave-mediated supercritical ethanol conditions, while preserving the nutrients and other valuable components in the algae. This single-step process can simultaneously and effectively extract the lipids from wet algae and transesterify them into crude biodiesel. Experimental runs were designed to optimize the process parameters and to evaluate their effects on algal biodiesel yield. The algal biomass characterization and algal biodiesel analysis were carried out by using various analytical instruments such as FTIR, SEM-EDS, TLC, GC-MS and transmission electron microscopy (TEM). The thermogravimetric analysis (TGA) under nitrogen and oxygen environments was also performed to examine the thermal and oxidative stability of ethyl esters produced from wet algae. This simple in situ transesterification process using a green solvent and catalyst-free approach can be a potentially efficient route for algal biodiesel production.

Theory and practice of a variable dome splitter for gas chromatography-olfactometry.

For olfactometric measurements in combination with gas chromatography a device is needed to split the GC effluent between the detector and the sniffing port. Fixed split ratios are obtained by simple flow splitters with appropriate restrictions towards the two outlets. Variable split ratios are possible with additional control flows. One such device is a dome splitter with one input flow (the GC effluent), two output flows (to the two outlets) and two control inputs. Preliminary experiments revealed deviations from the expected split ratios of such a device. The dimensioning of the flow restrictors at only one working point was not sufficient to obtain the expected split ratios over the whole temperature range of a GC run. Therefore a physical model of the flow system has been developed, taking into account the temperature dependence of the restrictors and the internal pressure in the dome. This included the solution of the flow (respectively the mass) balance under the condition of a compressible, isothermal and laminar flow regime. The measurements are in good agreement with theoretical calculations. The model can therefore be used to optimise the dimensions of the restrictions and to calculate the effective split ratio at a given temperature during the GC run.

Nannochloropsis production metrics in a scalable outdoor photobioreactor for commercial applications.

Commercial production of renewable energy feedstocks from microalgae will require reliable and scalable growth systems. Two and one half years of biomass and lipid productivity data were obtained with an industrial-scale outdoor photobioreactor operated in Fort Collins, Colorado (USA). The annualized volumetric growth rates for Nannochloropsis oculata (CCMP 525) and Nannochloropsis salina (CCMP 1776) were 0.16 g L(-1) d(-1) (peak=0.37 g L(-1) d(-1)) and 0.15 g L(-1) d(-1) (peak=0.37 g L(-1) d(-1)) respectively. The collective average lipid production was 10.7 m3 ha(-1) yr(-1) with a peak value of 36.3 m3 ha(-1) yr(-1). Results from this study are unique based on publication of biomass and corresponding lipid content combined with demonstration of energy savings realized through analysis of gas delivery requirements, water recycling from successive harvests with no effect on productivity, and culture stability through serial batch lineage data and chemotaxonomic analysis of fatty acid contents.

Optimization of microwave-assisted transesterification of dry algal biomass using response surface methodology.

The effect of microwave irradiation on the simultaneous extraction and transesterification (in situ transesterification) of dry algal biomass to biodiesel was investigated. A high degree of oil/lipid extraction from dry algal biomass and an efficient conversion of the oils/lipids to biodiesel were demonstrated in a set of well-designed experimental runs. A response surface methodology (RSM) was used to analyze the influence of the process variables (dry algae to methanol (wt/vol) ratio, catalyst concentration, and reaction time) on the fatty acid methyl ester conversion. Based on the experimental results and RSM analysis, the optimal conditions for this process were determined as: dry algae to methanol (wt/vol) ratio of around 1:12, catalyst concentration about 2 wt.%, and reaction time of 4 min. The algal biodiesel samples were analyzed with GC-MS and thin layer chromatography (TLC) methods. Transmission electron microscopy (TEM) images of the algal biomass samples before and after the extraction/transesterification reaction are also presented.

Regulation of the nitrogen transfer pathway in the arbuscular mycorrhizal symbiosis: gene characterization and the coordination of expression with nitrogen flux.

The arbuscular mycorrhiza (AM) brings together the roots of over 80% of land plant species and fungi of the phylum Glomeromycota and greatly benefits plants through improved uptake of mineral nutrients. AM fungi can take up both nitrate and ammonium from the soil and transfer nitrogen (N) to host roots in nutritionally substantial quantities. The current model of N handling in the AM symbiosis includes the synthesis of arginine in the extraradical mycelium and the transfer of arginine to the intraradical mycelium, where it is broken down to release N for transfer to the host plant. To understand the mechanisms and regulation of N transfer from the fungus to the plant, 11 fungal genes putatively involved in the pathway were identified from Glomus intraradices, and for six of them the full-length coding sequence was functionally characterized by yeast complementation. Two glutamine synthetase isoforms were found to have different substrate affinities and expression patterns, suggesting different roles in N assimilation. The spatial and temporal expression of plant and fungal N metabolism genes were followed after nitrate was added to the extraradical mycelium under N-limited growth conditions using hairy root cultures. In parallel experiments with (15)N, the levels and labeling of free amino acids were measured to follow transport and metabolism. The gene expression pattern and profiling of metabolites involved in the N pathway support the idea that the rapid uptake, translocation, and transfer of N by the fungus successively trigger metabolic gene expression responses in the extraradical mycelium, intraradical mycelium, and host plant.