Method for Investigation of Photobiological Effects of Light on Human Skin Cells Mediated by Low Doses of Light.

Driven by evolution, human skin cells have developed an extraordinary ability both to sense and to respond to the photons of sunlight through a plethora of photobiological interactions, activating intracellular signalling cascades and regulating skin cells homeostasis. It has recently been reported that some of these photobiological responses triggered by low levels of light (or the so-called photobiomodulation) could initiate beneficial therapeutic effects. Identification of these effective light-based therapeutic solutions requires in-depth understanding of the parameter space. The physical, biological, and chemical conditions that need to be fulfilled to facilitate such positive photobiological effects are to be carefully deciphered. Here, we provide the protocols that were specifically developed to investigate multidimensional parameter space driving photobiological interactions triggered by light (photobiomodulation) in the skin cells. The approach is based on the so-called design of experiment (DoE), a statistical method, which allows for the investigation of multidimensional parameters landscapes. This goes hand in hand with sharing practical tips for the design of light-based devices inducing these effects. To exemplify practical applications of the developed methods and light-based devices, we disclose experimental data sets and emphasize robustness and reproducibility of the results.

Pulsatile illumination for photobiology and optogenetics.

Living organisms exhibit a wide range of intrinsic adaptive responses to incident light. Likewise, in optogenetics, biological systems are tailored to initiate predetermined cellular processes upon light exposure. As genetically encoded, light-gated actuators, sensory photoreceptors are at the heart of these responses in both the natural and engineered scenarios. Upon light absorption, photoreceptors enter a series of generally rapid photochemical reactions leading to population of the light-adapted signaling state of the receptor. Notably, this state persists for a while before thermally reverting to the original dark-adapted resting state. As a corollary, the inactivation of photosensitive biological circuits upon light withdrawal can exhibit substantial inertia. Intermittent illumination of suitable pulse frequency can hence maintain the photoreceptor in its light-adapted state while greatly reducing overall light dose, thereby mitigating adverse side effects. Moreover, several photoreceptor systems may be actuated sequentially with a single light color if they sufficiently differ in their inactivation kinetics. Here, we detail the construction of programmable illumination devices for the rapid and parallelized testing of biological responses to diverse lighting regimes. As the technology is based on open electronics and readily available, inexpensive components, it can be adopted by most laboratories at moderate expenditure. As we exemplify for two use cases, the programmable devices enable the facile interrogation of diverse illumination paradigms and their application in optogenetics and photobiology.

An open-hardware platform for optogenetics and photobiology.

In optogenetics, researchers use light and genetically encoded photoreceptors to control biological processes with unmatched precision. However, outside of neuroscience, the impact of optogenetics has been limited by a lack of user-friendly, flexible, accessible hardware. Here, we engineer the Light Plate Apparatus (LPA), a device that can deliver two independent 310 to 1550 nm light signals to each well of a 24-well plate with intensity control over three orders of magnitude and millisecond resolution. Signals are programmed using an intuitive web tool named Iris. All components can be purchased for under $400 and the device can be assembled and calibrated by a non-expert in one day. We use the LPA to precisely control gene expression from blue, green, and red light responsive optogenetic tools in bacteria, yeast, and mammalian cells and simplify the entrainment of cyanobacterial circadian rhythm. The LPA dramatically reduces the entry barrier to optogenetics and photobiology experiments.

Analysis of Light Transport Features in Stone Fruits Using Monte Carlo Simulation.

The propagation of light in stone fruit tissue was modeled using the Monte Carlo (MC) method. Peaches were used as the representative model of stone fruits. The effects of the fruit core and the skin on light transport features in the peaches were assessed. It is suggested that the skin, flesh and core should be separately considered with different parameters to accurately simulate light propagation in intact stone fruit. The detection efficiency was evaluated by the percentage of effective photons and the detection sensitivity of the flesh tissue. The fruit skin decreases the detection efficiency, especially in the region close to the incident point. The choices of the source-detector distance, detection angle and source intensity were discussed. Accurate MC simulations may result in better insight into light propagation in stone fruit and aid in achieving the optimal fruit quality inspection without extensive experimental measurements.

Upgrading a microplate reader for photobiology and all-optical experiments.

Automation can vastly reduce the cost of experimental labor and thus facilitate high experimental throughput, but little off-the-shelf hardware for the automation of illumination experiments is commercially available. Here, we use inexpensive open-source electronics to add programmable illumination capabilities to a multimode microplate reader. We deploy this setup to characterize light-triggered phenomena in three different sensory photoreceptors. First, we study the photoactivation of Arabidopsis thaliana phytochrome B by light of different wavelengths. Second, we investigate the dark-state recovery kinetics of the Synechocystis sp. blue-light sensor Slr1694 at multiple temperatures and imidazole concentrations; while the kinetics of the W91F mutant of Slr1694 are strongly accelerated by imidazole, the wild-type protein is hardly affected. Third, we determine the light response of the Beggiatoa sp. photoactivatable adenylate cyclase bPAC in Chinese hamster ovary cells. bPAC is activated by blue light in dose-dependent manner with a half-maximal intensity of 0.58 mW cm(-2); intracellular cAMP spikes generated upon bPAC activation decay with a half time of about 5 minutes after light switch-off. Taken together, we present a setup which is easily assembled and which thus offers a facile approach to conducting illumination experiments at high throughput, reproducibility and fidelity.

Of ion pumps, sensors and channels - perspectives on microbial rhodopsins between science and history.

We present a historical overview of research on microbial rhodopsins ranging from the 1960s to the present date. Bacteriorhodopsin (BR), the first identified microbial rhodopsin, was discovered in the context of cell and membrane biology and shown to be an outward directed proton transporter. In the 1970s, BR had a big impact on membrane structural research and bioenergetics, that made it to a model for membrane proteins and established it as a probe for the introduction of various biophysical techniques that are widely used today. Halorhodopsin (HR), which supports BR physiologically by transporting negatively charged Cl⁻ into the cell, is researched within the microbial rhodopsin community since the late 1970s. A few years earlier, the observation of phototactic responses in halobacteria initiated research on what are known today as sensory rhodopsins (SR). The discovery of the light-driven ion channel, channelrhodopsin (ChR), serving as photoreceptors for behavioral responses in green alga has complemented inquiries into this photoreceptor family. Comparing the discovery stories, we show that these followed quite different patterns, albeit the objects of research being very similar. The stories of microbial rhodopsins present a comprehensive perspective on what can nowadays be considered one of nature's paradigms for interactions between organisms and light. Moreover, they illustrate the unfolding of this paradigm within the broader conceptual and instrumental framework of the molecular life sciences. This article is part of a Special Issue entitled: Retinal Proteins - You can teach an old dog new tricks.

Study of idiopathic, exogenous photodermatoses. Part 1: pathophysiology and technical aspects of photobiologic studies.

Photodermatoses are skin conditions that are induced or exacerbated by electromagnetic radiation (including visible light, UV light, and infrared radiation) from the sun or artificial light sources. In Part 1 of this series we review current understanding of the pathophysiology of these processes and their classification. We also discuss technical aspects and the basic physics of photobiology and describe the equipment required for photobiologic testing and calibration (light sources and measurement instruments).

Hydrogen photoproduction by Rhodopseudomonas palustris 42OL cultured at high irradiance under a semicontinuous regime.

The main goal of this study was to increase the hydrogen production rate improving the culture technique and the photobioreactor performances. Experiments were carried out at a constant culture temperature of 30°C and at an average irradiance of 480 W m(-2) using a cylindrical photobioreactor (4.0 cm, internal diameter). The culture technique, namely, the semicontinuous regime for growing Rhodopseudomonas palustris 42OL made it possible to achieve a very high daily hydrogen production rate of 594 ± 61 mL (H(2)) L(-1) d(-1). This value, never reported for this strain, corresponds to about 25 mL (H(2)) L(-1) h(-1), and it was obtained when the hydraulic retention time (HRT) was of 225 hours. Under the same growth conditions, a very high biomass production rate (496 ± 45 mg (dw) L(-1) d(-1)) was also achieved. Higher or lower HRTs caused a reduction in both the hydrogen and the biomass production rates. The malic-acid removal efficiency (MA(re)) was always higher than 90%. The maximal hydrogen yield was 3.03 mol H(2) mol MA(-1) at the HRT of 360 hours. The highest total energy conversion efficiency was achieved at the HRT of 225 hours.

Integrating photobiological hydrogen production with dye-metal bioremoval from simulated textile wastewater.

The study reports production of hydrogen in photobioreactors with free (PBR(Fr)) and immobilized (PBR(Imm)) Nostoc biomass at enhanced and sustained rates. Before running the photobioreactors, effects of different immobilization matrices and cyanobacterial dose on hydrogen production were studied in batch mode. As hydrogen production in the PBRs declined spent biomass from the photobioreactors were collected and utilized further for column biosorption of highly toxic dyes (Reactive Red 198+Crystal Violet) and metals (hexavalent chromium and bivalent cobalt) from simulated textile wastewater. Breakthrough time, adsorption capacity and exhaustion time of the biosorption column were studied. The photobioreactors with free and immobilized cyanobacterium produced hydrogen at average rates of 101 and 151 µmol/h/mg Chl a, respectively over 15 days, while the adsorption capacity of the spent biomass was up to 1.4 and 0.23 mg/g for metals and 15 and 1.75 mg/g for the dyes, respectively in continuous column mode.

Optical microplates for photonic high throughput screening of algal photosynthesis and biofuel production.

Biological systems respond not only to chemical stimuli (drugs, proteins) but also to physical stimuli (light, heat, stress). Though there are many high throughput tools for screening chemical stimuli, no such tool exists for screening of physical stimuli. This paper presents a novel instrument for photonic high throughput screening of photosynthesis, a light-driven bioprocess. The optical microplate has a footprint identical to a standard 96 well plate, and it provides temporal and intensity control of light in each individual well. Intensity control provides 128 dimming levels (7-bit resolution), with maximum intensity 120 mE/cm(2). Temporal modulation, used for studying dynamics and regulation of photosynthesis, can be as low as 10 µs. We used photonic screening for high throughput studies of algal growth rates and photosynthetic efficiency, using the model organism Dunaliella tertiolecta, a lipid producing algae of interest in biofuel production. Due to the ability to conduct 96 studies in parallel, experiments that would require 2 years using conventional tools can be completed in 1 week. This instrument opens up novel high throughput protocols for photobiology and the growing field of phenomics.

Intimate coupling of photocatalysis and biodegradation in a photocatalytic circulating-bed biofilm reactor.

Coupling advanced oxidative pretreatment with subsequent biodegradation demonstrates potential for treating wastewaters containing biorecalcitrant and inhibitory organic constituents. However, advanced oxidation is indiscriminate, producing a range of products that can be too oxidized, unavailable for biodegradation, or toxic themselves. This problem could be overcome if advanced oxidation and biodegradation occurred together, an orientation called intimate coupling; then, biodegradable organics are removed as they are formed, focusing the chemical oxidant on the non-biodegradable fraction. Intimate coupling has seemed impossible because the conditions of advanced oxidation, for example, hydroxyl radicals and sometimes UV-light, are severely toxic to microorganisms. Here, we demonstrate that a novel photocatalytic circulating-bed biofilm reactor (PCBBR), which utilizes macro-porous carriers to protect biofilm from toxic reactants and UV light, achieves intimate coupling. We demonstrate the viability of the PCBBR system first with UV only and acetate, where the carriers grew biofilm and sustained acetate biodegradation despite continuous UV irradiation. Images obtained by scanning electron microscopy and confocal laser scanning microscopy show bacteria living behind the exposed surface of the cubes. Second, we used slurry-form Degussa P25 TiO2 to initiate photocatalysis of inhibitory 2,4,5-trichlorophenol (TCP) and acetate. With no bacterial carriers, photocatalysis and physical processes removed TCP and COD to 32% and 26% of their influent levels, but addition of biofilm carriers decreased residuals to 2% and 4%, respectively. Biodegradation alone could not remove TCP. Photomicrographs clearly show that biomass originally on the exterior of the carriers was oxidized (charred), but biofilm a short distance within the carriers was protected. Finally, we coated TiO2 directly onto the carrier surface, producing a hybrid photocatalytic-biological carrier. These carriers likewise demonstrated the concept of photocatalytic degradation of TCP coupled with biodegradation of acetate, but continued TCP degradation required augmentation with slurry-form TiO2.

A photobioreactor system for precision cultivation of photoautotrophic microorganisms and for high-content analysis of suspension dynamics.

Small-scale photobioreactors for cultivation of photoautotrophic microbes are required for precise characterization of the growth parameters of wild-type and engineered strains of these organisms, for their screening, and for optimization of culture conditions. Here, we describe the design and use of a flat-cuvette photobioreactor that allows accurate control of culture irradiance, temperature, pH, and gas composition combined with real-time monitoring by a built-in fluorometer and densitometer. The high-power LED light source generates precise irradiance levels that are programmed by user-designed protocols. The irradiance, temperature, and gas composition may be static or dynamically modulated, while optical density and pH may be stabilized in turbidostat and pH-stat modes, respectively. We demonstrate that the instrument is able to detect minute variations of growth caused, for example, by sudden dilution or by circadian rhythms. The sensitivity of the instrument is sufficient to monitor suspension optical density as low as 10(-2). This newly designed photobioreactor can significantly contribute to the study and use of photoautotrophic microbes in systems biology and biotechnology.

Design process of an area-efficient photobioreactor.

This article describes the design process of the Green Solar Collector (GSC), an area-efficient photobioreactor for the outdoor cultivation of microalgae. The overall goal has been to design a system in which all incident sunlight on the area covered by the reactor is delivered to the algae at such intensities that the light energy can be efficiently used for biomass formation. A statement of goals is formulated and constraints are specified to which the GSC needs to comply. Specifications are generated for a prototype which form and function achieve the stated goals and satisfy the specified constraints. This results in a design in which sunlight is captured into vertical plastic light guides. Sunlight reflects internally in the guide and eventually scatters out of the light guide into flat-panel photobioreactor compartments. Sunlight is focused on top of the light guides by dual-axis positioning of linear Fresnel lenses. The shape and material of the light guide is such that light is maintained in the guides when surrounded by air. The bottom part of a light guide is sandblasted to obtain a more uniform distribution of light inside the bioreactor compartment and is triangular shaped to ensure the efflux of all light out of the guide. Dimensions of the guide are such that light enters the flat-panel photobioreactor compartment at intensities that can be efficiently used by the biomass present. The integration of light capturing, transportation, distribution and usage is such that high biomass productivities per area can be achieved.

Simulations of light intensity variation in photobioreactors.

In photobioreactors, turbulent flow conditions and light gradients frequently occur. Thus, algal cells cultivated in such reactors experience fluctuations in light intensity. This work presents a new method for the calculation of these light-dark patterns. The investigation is focused on temporal and spatial aspects of light patterns which may affect the photosynthetic reaction. The method combines computational fluid dynamics simulations of three-dimensional turbulent single-phase fluid flow with statistical particle tracking and signal analysis. In this way, light-dark phases are derived which affect singular (algal) cells. An example case is presented of a tubular photobioreactor in which static mixers are used for the efficient mixing of liquid and also of gases with liquid. Particle trajectories representing the path of algal cells were analysed to obtain light fluctuations on single cells. Particles were exposed to light-dark phases with frequencies between 3 and 25Hz in a helical mixer at a mean velocity of 0.5ms(-1), which contrasts to the case of a tube without static mixers, where only frequencies of 0.2-3.1Hz were obtained under the same conditions. The simulations show the potential of improving radial flow in a tubular photobioreactor by means of using a static mixer and the usefulness of CFD and trajectory analysis for scale-down/scale-up.

Carbon dioxide fixation by Chlorella kessleri, C. vulgaris, Scenedesmus obliquus and Spirulina sp. cultivated in flasks and vertical tubular photobioreactors.

CO(2) at different concentrations were added to cultures of the eukaryotic microalgae, Chlorella kessleri, C. vulgaris and Scenedesmus obliquus, and the prokaryotic cyanobacterium, Spirulina sp., growing in flasks and in a photobioreactor. In each case, the best kinetics and carbon fixation rate were with a vertical tubular photobioreactor. Overall, Spirulina sp. had the highest rates. Spirulina sp., Sc. obliquus and C. vulgaris could grow with up to 18% CO(2).

In situ monitoring of cell concentration in a photobioreactor using image analysis: comparison of uniform light distribution model and artificial neural networks.

Light intensity is a very important factor that determines the growth of photosynthetic cells. In this study, the light distribution in a photobioreactor was analyzed by processing the images captured with a digital camera. The contour images obtained by filtering the original images clearly showed the effects of the cell concentration and external light intensity on the light distribution. Image-processing techniques were then applied to predict the cell density in the photobioreactor. To correlate the cell concentration with the light intensity in the photobioreactor, the captured images were processed using two different approaches. The first method involved the use of an average gray value after deriving a simplified model equation that could be related to the cell density. The second method involved the use of local points instead of a representative value. In this case, an artificial neural network model was adopted to infer the cell density from the information of the local points. By using these two methods, it was possible to relate the image data to the cell concentration. Finally, we compared these two methods with regard to their accuracy, easiness, and effectiveness.

Comparative analysis of the outdoor culture of Haematococcus pluvialis in tubular and bubble column photobioreactors.

The present paper makes a comparative analysis of the outdoor culture of H. pluvialis in a tubular photobioreactor and a bubble column. Both reactors had the same volume and were operated in the same way, thus allowing the influence of the reactor design to be analyzed. Due to the large changes in cell morphology and biochemical composition of H. pluvialis during outdoor culture, a new, faster methodology has been developed for their evaluation. Characterisation of the cultures is carried out on a macroscopic scale using a colorimetric method that allows the simultaneous determination of biomass concentration, and the chlorophyll, carotenoid and astaxanthin content of the biomass. On the microscopic scale, a method was developed based on the computer analysis of digital microscopic images. This method allows the quantification of cell population, average cell size and population homogeneity. The accuracy of the methods was verified during the operation of outdoor photobioreactors on a pilot plant scale. Data from the reactors showed tubular reactors to be more suitable for the production of H. pluvialis biomass and/or astaxanthin, due to their higher light availability. In the tubular photobioreactor biomass concentrations of 7.0 g/L (d.wt.) were reached after 16 days, with an overall biomass productivity of 0.41 g/L day. In the bubble column photobioreactor, on the other hand, the maximum biomass concentration reached was 1.4 g/L, with an overall biomass productivity of 0.06 g/L day. The maximum daily biomass productivity, 0.55 g/L day, was reached in the tubular photobioreactor for an average irradiance inside the culture of 130 microE/m2s. In addition, the carotenoid content of biomass from tubular photobioreactor increased up to 2.0%d.wt., whereas that of the biomass from the bubble column remained roughly constant at values of 0.5%d.wt. It should be noted that in the tubular photobioreactor under conditions of nitrate saturation, there was an accumulation of carotenoids due to the high irradiance in this reactor, their content in the biomass increasing from 0.5 to 1.0%d.wt. However, carotenoid accumulation mainly took place when nitrate concentration in the medium was below 5.0mM, conditions which were only observed in the tubular photobioreactor. A similar behaviour was observed for astaxanthin, with maximum values of 1.1 and 0.2%d.wt. measured in the tubular and bubble column photobioreactors, respectively. From these data astaxanthin productivities of 4.4 and 0.12 mg/L day were calculated for the tubular and the bubble column photobioreactors. Accumulation of carotenoids was also accompanied by an increase in cell size from 20 to 35 microm, which was only observed in the tubular photobioreactors. Thus it may be concluded that the methodology developed in the present study allows the monitoring of H. pluvialis cultures characterized by fast variations of cell morphology and biochemical composition, especially in outdoor conditions, and that tubular photobioreactors are preferable to bubble columns for the production of biomass and/or astaxanthin.

A fully predictive model for one-dimensional light attenuation by Chlamydomonas reinhardtii in a torus photobioreactor.

The light attenuation in a photobioreactor is determined using a fully predictive model. The optical properties were first calculated, using a data bank of the literature, from only the knowledge of pigments content, shape, and size distributions of cultivated cells which are a function of the physiology of the current species. The radiative properties of the biological turbid medium were then deduced using the exact Lorenz-Mie theory. This method is experimentally validated using a large-size integrating sphere photometer. The radiative properties are then used in a rectangular, one-dimensional two-flux model to predict radiant light attenuation in a photobioreactor, considering a quasi-collimated field of irradiance. Combination of this radiative model with the predictive determination of optical properties is finally validated by in situ measurement of attenuation profiles in a torus photobioreactor cultivating the microalgae Chlamydomonas reinhardtii, after a complete and proper characterization of the incident light flux provided by the experimental set-up.

Design, operation, and modeling of a membrane photobioreactor to study the growth of the Cyanobacterium Arthrospira platensis in space conditions.

A membrane photobioreactor was designed, implemented and used to grow the cyanobacterium Arthrospira platensis PCC 8005 in batch mode. Growth was followed directly by monitoring optical density and indirectly by measuring pressure increase due to the oxygen produced and separated from the liquid phase by diffusion through a hydrophobic membrane, and pH increase due to carbon consumption. When the pressure attained an upper limit, valves opened automatically, and the oxygen in the gas chamber was flushed out with nitrogen. As expected, two growth phases were observed, a short exponential phase followed by a linear phase, indicating limitation by light transfer. Growth rate during the second phase was measured easily and accurately, and consistency of optical density, pressure and pH data values was checked using a model of the system. Pressure measurement was found best suited to monitoring and measuring growth rate in space in terms of accuracy, precision and reliability.