On-line stable isotope gas exchange reveals an inducible but leaky carbon concentrating mechanism in Nannochloropsis salina.

Carbon concentrating mechanisms (CCMs) are common among microalgae, but their regulation and even existence in some of the most promising biofuel production strains is poorly understood. This is partly because screening for new strains does not commonly include assessment of CCM function or regulation despite its fundamental role in primary carbon metabolism. In addition, the inducible nature of many microalgal CCMs means that environmental conditions should be considered when assessing CCM function and its potential impact on biofuels. In this study, we address the effect of environmental conditions by combining novel, high frequency, on-line (13)CO2 gas exchange screen with microscope-based lipid characterization to assess CCM function in Nannochloropsis salina and its interaction with lipid production. Regulation of CCM function was explored by changing the concentration of CO2 provided to continuous cultures in airlift bioreactors where cell density was kept constant across conditions by controlling the rate of media supply. Our isotopic gas exchange results were consistent with N. salina having an inducible "pump-leak" style CCM similar to that of Nannochloropsis gaditana. Though cells grew faster at high CO2 and had higher rates of net CO2 uptake, we did not observe significant differences in lipid content between conditions. Since the rate of CO2 supply was much higher for the high CO2 conditions, we calculated that growing cells bubbled with low CO2 is about 40 % more efficient for carbon capture than bubbling with high CO2. We attribute this higher efficiency to the activity of a CCM under low CO2 conditions.

Host cell pigmentation in Scenedesmus dimorphus as a beacon for nascent parasite infection.

Biofuels derived from the mass cultivation of algae represent an emerging industry that aims to partially displace petroleum based fuels. Outdoor, open-pond, and raceway production facilities are attractive options for the mass culture of algae however, this mode of cultivation leaves the algae susceptible to epidemics from a variety of environmental challenges. Infestations can result in complete collapse of the algal populations and destruction of their valuable products making it paramount to understand the host-pathogen relationships of known algal pests in order to develop mitigation strategies. In the present work, we characterize the spatial-temporal response of photosynthetic pigments in Scenedesmus dimorphus to infection from Amoeboaphelidium protococcarum, a destructive endoparasite, with the goal of understanding the potential for early detection of infection via host pigment changes. We employed a hyperspectral confocal fluorescence microscope to quantify these changes in pigmentation with high spatial and spectral resolution during early parasite infection. Carotenoid abundance and autofluorescence increased within the first 24 h of infection while chlorophyll emission remained constant. Changes in host cell photosynthesis and bulk chlorophyll content were found to lag behind parasite replication. The results herein raise the possibility of using host-cell pigment changes as indicators of nascent parasite infection.

Photosynthetic pigment localization and thylakoid membrane morphology are altered in Synechocystis 6803 phycobilisome mutants.

Cyanobacteria are oxygenic photosynthetic prokaryotes that are the progenitors of the chloroplasts of algae and plants. These organisms harvest light using large membrane-extrinsic phycobilisome antenna in addition to membrane-bound chlorophyll-containing proteins. Similar to eukaryotic photosynthetic organisms, cyanobacteria possess thylakoid membranes that house photosystem (PS) I and PSII, which drive the oxidation of water and the reduction of NADP+, respectively. While thylakoid morphology has been studied in some strains of cyanobacteria, the global distribution of PSI and PSII within the thylakoid membrane and the corresponding location of the light-harvesting phycobilisomes are not known in detail, and such information is required to understand the functioning of cyanobacterial photosynthesis on a larger scale. Here, we have addressed this question using a combination of electron microscopy and hyperspectral confocal fluorescence microscopy in wild-type Synechocystis species PCC 6803 and a series of mutants in which phycobilisomes are progressively truncated. We show that as the phycobilisome antenna is diminished, large-scale changes in thylakoid morphology are observed, accompanied by increased physical segregation of the two photosystems. Finally, we quantified the emission intensities originating from the two photosystems in vivo on a per cell basis to show that the PSI:PSII ratio is progressively decreased in the mutants. This results from both an increase in the amount of photosystem II and a decrease in the photosystem I concentration. We propose that these changes are an adaptive strategy that allows cells to balance the light absorption capabilities of photosystems I and II under light-limiting conditions.

Algal biomass constituent analysis: method uncertainties and investigation of the underlying measuring chemistries.

Algal biomass compositional analysis data form the basis of a large number of techno-economic process analysis models that are used to investigate and compare different processes in algal biofuels production. However, the analytical methods used to generate these data are far from standardized. This work investigated the applicability of common methods for rapid chemical analysis of biomass samples with respect to accuracy and precision. This study measured lipids, protein, carbohydrates, ash, and moisture of a single algal biomass sample at 3 institutions by 8 independent researchers over 12 separate workdays. Results show statistically significant differences in the results from a given analytical method among laboratories but not between analysts at individual laboratories, suggesting consistent training is a critical issue for empirical analytical methods. Significantly different results from multiple lipid and protein measurements were found to be due to different measurement chemistries. We identified a set of compositional analysis procedures that are in best agreement with data obtained by more advanced analytical procedures. The methods described here and used for the round robin experiment do not require specialized instrumentation, and with detailed analytical documentation, the differences between laboratories can be markedly reduced.

Physiological effects of free fatty acid production in genetically engineered Synechococcus elongatus PCC 7942.

The direct conversion of carbon dioxide into biofuels by photosynthetic microorganisms is a promising alternative energy solution. In this study, a model cyanobacterium, Synechococcus elongatus PCC 7942, is engineered to produce free fatty acids (FFA), potential biodiesel precursors, via gene knockout of the FFA-recycling acyl-ACP synthetase and expression of a thioesterase for release of the FFA. Similar to previous efforts, the engineered strains produce and excrete FFA, but the yields are too low for large-scale production. While other efforts have applied additional metabolic engineering strategies in an attempt to boost FFA production, we focus on characterizing the engineered strains to identify the physiological effects that limit cell growth and FFA synthesis. The strains engineered for FFA-production show reduced photosynthetic yields, chlorophyll-a degradation, and changes in the cellular localization of the light-harvesting pigments, phycocyanin and allophycocyanin. Possible causes of these physiological effects are also identified. The addition of exogenous linolenic acid, a polyunsaturated FFA, to cultures of S. elongatus 7942 yielded a physiological response similar to that observed in the FFA-producing strains with only one notable difference. In addition, the lipid constituents of the cell and thylakoid membranes in the FFA-producing strains show changes in both the relative amounts of lipid components and the degree of saturation of the fatty acid side chains. These changes in lipid composition may affect membrane integrity and structure, the binding and diffusion of phycobilisomes, and the activity of membrane-bound enzymes including those involved in photosynthesis. Thus, the toxicity of unsaturated FFA and changes in membrane composition may be responsible for the physiological effects observed in FFA-producing S. elongatus 7942. These issues must be addressed to enable the high yields of FFA synthesis necessary for large-scale biofuel production.

Multiplex fluorometric assessment of nutrient limitation as a strategy for enhanced lipid enrichment and harvesting of Neochloris oleoabundans.

Detailed in this study are the results of fluorometric assays used to assess the impact of gradual nutrient limitation versus punctuated nitrate limitation on the lipid content and morphology of Neochloris oleoabundans cells in batch culture. Punctuated nitrate limitation was imposed during pre-log, log, late-log, stationary, and senescent growth phases, and the cells were analyzed by bulk fluorescence emission, flow cytometry, and hyperspectral fluorescence imaging. In addition to intrinsic spectroscopic signatures provided by scatter and endogenous fluorescence, Nile Red staining was employed to monitor relative changes in lipid concentration. Analysis of the fluorescence images and temporal data sets was performed using multivariate curve resolution and fitting to logistic growth models to extract parameters of interest. The spectral components independently isolated from the image and temporal data sets showed close agreement with one another, especially relating to chlorophylls and Nile Red in polar and neutral lipid fractions, respectively. The fastest accumulation and highest total neutral lipid per cell and per chlorophyll were obtained with punctuated nitrate limitation during log phase growth on day 4 of culture. The presence of unbound chlorophyll in the resulting lipid bodies supports a membrane recycling TAG accumulation mechanism mediated by chloropolast-ER lipid exchange. Furthermore, an increase in cell size, indicated by forward scatter, was also found to correlate with increased neutral lipid, providing a size selection mechanism for passive harvest of algal cells at peak lipid enrichment.

In vivo hyperspectral confocal fluorescence imaging to determine pigment localization and distribution in cyanobacterial cells.

Hyperspectral confocal fluorescence imaging provides the opportunity to obtain individual fluorescence emission spectra in small ( approximately 0.03-microm(3)) volumes. Using multivariate curve resolution, individual fluorescence components can be resolved, and their intensities can be calculated. Here we localize, in vivo, photosynthesis-related pigments (chlorophylls, phycobilins, and carotenoids) in wild-type and mutant cells of the cyanobacterium Synechocystis sp. PCC 6803. Cells were excited at 488 nm, exciting primarily phycobilins and carotenoids. Fluorescence from phycocyanin, allophycocyanin, allophycocyanin-B/terminal emitter, and chlorophyll a was resolved. Moreover, resonance-enhanced Raman signals and very weak fluorescence from carotenoids were observed. Phycobilin emission was most intense along the periphery of the cell whereas chlorophyll fluorescence was distributed more evenly throughout the cell, suggesting that fluorescing phycobilisomes are more prevalent along the outer thylakoids. Carotenoids were prevalent in the cell wall and also were present in thylakoids. Two chlorophyll fluorescence components were resolved: the short-wavelength component originates primarily from photosystem II and is most intense near the periphery of the cell; and the long-wavelength component that is attributed to photosystem I because it disappears in mutants lacking this photosystem is of higher relative intensity toward the inner rings of the thylakoids. Together, the results suggest compositional heterogeneity between thylakoid rings, with the inner thylakoids enriched in photosystem I. In cells depleted in chlorophyll, the amount of both chlorophyll emission components was decreased, confirming the accuracy of the spectral assignments. These results show that hyperspectral fluorescence imaging can provide unique information regarding pigment organization and localization in the cell.

Accurate detection of low levels of fluorescence emission in autofluorescent background: francisella-infected macrophage cells.

Cellular autofluorescence, though ubiquitous when imaging cells and tissues, is often assumed to be small in comparison to the signal of interest. Uniform estimates of autofluorescence intensity obtained from separate control specimens are commonly employed to correct for autofluorescence. While these may be sufficient for high signal-to-background applications, improvements in detector and probe technologies and introduction of spectral imaging microscopes have increased the sensitivity of fluorescence imaging methods, exposing the possibility of effectively probing the low signal-to-background regime. With spectral imaging, reliable monitoring of signals near or even below the noise levels of the microscope is possible if compensation for autofluorescence and background signals can be performed accurately. We demonstrate the importance of accurate autofluorescence modeling and the utility of spectral imaging and multivariate analysis methods using a case study focusing on fluorescence confocal spectral imaging of host-pathogen interactions. In this application fluorescent proteins are produced when Francisella novicida invade host macrophage cells. The resulting analyte signal is spectrally overlapped and typically weaker than the cellular autofluorescence. In addition to discussing the advantages of spectral imaging for following pathogen invasion, we present the spectral properties and cellular origin of macrophage autofluorescence.

Antimicrobial peptide interactions with silica bead supported bilayers and E. coli: buforin II, magainin II, and arenicin.

Using the unique quantitative capabilities of hyperspectral confocal microscopy combined with multivariate curve resolution, a comparative approach was employed to gain a deeper understanding of the different types of interactions of antimicrobial peptides (AMPs) with biological membranes and cellular compartments. This approach allowed direct comparison of the dynamics and local effects of buforin II, magainin II, and arenicin with nanoporous silica bead supported bilayers and living E. coli. Correlating between experiments and comparing these responses have yielded several important discoveries for pursuing the underlying biophysics of bacteriocidal specificity and the connection between structure and function in various cellular environments. First, a novel fluorescence method for direct comparison of a model and living system is demonstrated by utilizing the membrane partitioning and environmental sensitivity of propidium iodide. Second, measurements are presented comparing the temporal dynamics and local equilibrium concentrations of the different antimicrobial agents in the membrane and internal matrix of the described systems. Finally, we discuss how the data lead to a deeper understanding of the roles of membrane penetration and permeabilization in the action of these AMPs.

Hyperspectral confocal fluorescence imaging: exploring alternative multivariate curve resolution approaches.

Hyperspectral confocal fluorescence microscopy, when combined with multivariate curve resolution (MCR), provides a powerful new tool for improved quantitative imaging of multi-fluorophore samples. Generally, fully non-negatively constrained models are used in the constrained alternating least squares MCR analyses of hyperspectral images since real emission components are expected to have non-negative pure emission spectra and concentrations. However, in this paper, we demonstrate four separate cases in which partially constrained models are preferred over the fully constrained MCR models. These partially constrained MCR models can sometimes be preferred when system artifacts are present in the data or where small perturbations of the major emission components are present due to environmental effects or small geometric changes in the fluorescing species. Here we demonstrate that in the cases of hyperspectral images obtained from multicomponent spherical beads, autofluorescence from fixed lung epithelial cells, fluorescence of quantum dots in aqueous solutions, and images of mercurochrome-stained endosperm portions of a wild-type corn seed, these alternative, partially constrained MCR analyses provide improved interpretability of the MCR solutions. Often the system artifacts or environmental effects are more readily described as first and/or second derivatives of the main emission components in these alternative MCR solutions since they indicate spectral shifts and/or spectral broadening or narrowing of the emission bands, respectively. Thus, this paper serves to demonstrate the need to test alternative partially constrained models when analyzing hyperspectral images with MCR methods.

The role of photo-osmotic adaptation in semi-continuous culture and lipid particle release from Dunaliella viridis.

Although great efforts have been made to elucidate the phenotypic responses of alga to varying levels of nutrients, osmotic environments, and photosynthetically active radiation intensities, the role of interactions among these variables is largely nebulous. Here, we describe a general method for establishing and maintaining semi-continuous cultures of the halophilic microalgal production strain, Dunaliella viridis, that is independent of variations in salinity and illumination intensity. Using this method, the cultures were evaluated to elucidate the overlapping roles of photosynthetic and osmotic adaptation on the accumulation and compositional variation of the biomass, photosynthetic productivity, and physiological biomarkers, as well as spectroscopic and morphological details at the single-cell level. Correlation matrices defining the relationships among the observables and based on variation of the illumination intensity and salinity were constructed for predicting bioproduct yields for varying culture conditions. Following maintenance of stable cultures for 6-week intervals, phenotypic responses to photo-osmotic drift were explored using a combination of single-cell hyperspectral fluorescence imaging and flow cytometry. In addition to morphological changes, release of lipid microparticles from the cells that is disproportionate to cell lysis was observed under hypotonic drift, indicating the existence of a reversible membrane permeation mechanism in Dunaliella. This phenomenon introduces the potential for low-cost strategies for recovering lipids and pigments from the microalgae by minimizing the requirement for energy intensive harvesting and dewatering of the biomass. The results should be applicable to outdoor culture, where seasonal changes resulting in variable solar flux and precipitation and evaporation rates are anticipated.

Carotenoid distribution in living cells of Haematococcus pluvialis (Chlorophyceae).

Haematococcus pluvialis is a freshwater unicellular green microalga belonging to the class Chlorophyceae and is of commercial interest for its ability to accumulate massive amounts of the red ketocarotenoid astaxanthin (3,3'-dihydroxy-ß,ß-carotene-4,4'-dione). Using confocal Raman microscopy and multivariate analysis, we demonstrate the ability to spectrally resolve resonance-enhanced Raman signatures associated with astaxanthin and ß-carotene along with chlorophyll fluorescence. By mathematically isolating these spectral signatures, in turn, it is possible to locate these species independent of each other in living cells of H. pluvialis in various stages of the life cycle. Chlorophyll emission was found only in the chloroplast whereas astaxanthin was identified within globular and punctate regions of the cytoplasmic space. Moreover, we found evidence for ß-carotene to be co-located with both the chloroplast and astaxanthin in the cytosol. These observations imply that ß-carotene is a precursor for astaxanthin and the synthesis of astaxanthin occurs outside the chloroplast. Our work demonstrates the broad utility of confocal Raman microscopy to resolve spectral signatures of highly similar chromophores in living cells.

Systematic analysis of micromixers to minimize biofouling on reverse osmosis membranes.

Micromixers, UV-curable epoxy traces printed on the surface of a reverse osmosis membrane, were tested on a cross-flow system to determine their success at reducing biofouling. Biofouling was quantified by measuring the rate of permeate flux decline and the median bacteria concentration on the surface of the membrane (as determined by fluorescence intensity counts due to nucleic acid stains as measured by hyperspectral imaging). The micromixers do not appear to significantly increase the pressure needed to maintain the same initial permeate flux and salt rejection. Chevrons helped prevent biofouling of the membranes in comparison with blank membranes. The chevron design controlled where the bacteria adhered to the membrane surface. However, blank membranes with spacers had a lower rate of permeate flux decline than the membranes with chevrons despite having greater bacteria concentrations on their surfaces. With better optimization of the micromixer design, the micromixers could be used to control where the bacteria will adhere to the surface and create a more biofouling resistant membrane that will help to drive down the cost of water treatment.

Cell-directed integration into three-dimensional lipid-silica nanostructured matrices.

We report a unique approach in which living cells direct their integration into 3D solid-state nanostructures. Yeast cells deposited on a weakly condensed lipid/silica thin film mesophase actively reconstruct the surface to create a fully 3D bio/nano interface, composed of localized lipid bilayers enveloped by a lipid/silica mesophase, through a self-catalyzed silica condensation process. Remarkably, this integration process selects exclusively for living cells over the corresponding apoptotic cells (those undergoing programmed cell death), via the development of a pH gradient, which catalyzes silica deposition and the formation of a coherent interface between the cell and surrounding silica matrix. Added long-chain lipids or auxiliary nanocomponents are localized within the pH gradient, allowing the development of complex active and accessible bio/nano interfaces not achievable by other synthetic methods. Overall, this approach provides the first demonstration of active cell-directed integration into a nominally solid-state three-dimensional architecture. It promises a new means to integrate "bio" with "nano" into platforms useful to study and manipulate cellular behavior at the individual cell level and to interface living organisms with electronics, photonics, and fluidics.

Hyperspectral confocal microscope.

We have developed a new, high performance, hyperspectral microscope for biological and other applications. For each voxel within a three-dimensional specimen, the microscope simultaneously records the emission spectrum from 500 nm to 800 nm, with better than 3 nm spectral resolution. The microscope features a fully confocal design to ensure high spatial resolution and high quality optical sectioning. Optical throughput and detection efficiency are maximized through the use of a custom prism spectrometer and a backside thinned electron multiplying charge coupled device (EMCCD) array. A custom readout mode and synchronization scheme enable 512-point spectra to be recorded at a rate of 8300 spectra per second. In addition, the EMCCD readout mode eliminates curvature and keystone artifacts that often plague spectral imaging systems. The architecture of the new microscope is described in detail, and hyperspectral images from several specimens are presented.