Microalgae-derived hydrogen production towards low carbon emissions via large-scale outdoor systems.

Hydrogen as a clean fuel is receiving attention because it generates only water and a small amount of nitrogen oxide upon combustion. Biohydrogen production using microalgae is considered to be a highly promising carbon-neutral technology because it can secure renewable energy while efficiently reducing CO2 emissions. However, previous studies have mainly focused on improving the biological performance of microalgae; these approaches have struggled to achieve breakthroughs in commercialization because they do not heavily consider the complexity of the entire production process with microalgae, including large-scale cultivation, biomass harvest, and biomass storage. This work presents an in-depth analysis of the state-of-the-art technologies focused on large-scale cultivation systems with efficient downstream processes. Considering the individual processes of biohydrogen production, strategies are discussed to minimize carbon emissions and improve productivity simultaneously. A comprehensive understanding of microalgae-derived biohydrogen production suggests future directions for realizing environmental and economic sustainability.

Development of a limitless scale-up photobioreactor for highly efficient photosynthesis-based polyhydroxybutyrate (PHB)-producing cyanobacteria.

Photosynthetic polyhydroxybutyrate (PHB) production is an attractive technology for realizing a sustainable society by simultaneously producing useful biodegradable plastics and mitigating CO2. It is necessary to establish an economical large-scale photobioreactor (PBR) capable of effectively cultivating photosynthetic microorganisms such as cyanobacteria. A roll-to-roll winding machine/heat-sealer hybrid system for fabricating an easy-to-scale-up PBR was developed in the present study. The baffle design was optimized to facilitate mass transfer within the PBR, and the operating conditions of the gas sparger were investigated to maximize the CO2 transfer efficiency. The newly developed PBR was able to produce biomass of PHB content 10.7 w/w% at a rate of 6.861 g m-2 d-1, 21 % improved biomass productivity compared with the existing PBR. It was confirmed that biomass productivity was maintained even when PBR was scaled up to 2 tons. Consequently, the newly developed PBR is expected to improve the feasibility of photosynthetic PHB production.

Kinetic analysis of microalgae cultivation utilizing 3D-printed real-time monitoring system reveals potential of biological CO2 conversion.

The microalgae-based bioconversion process is a promising carbon utilization technology because it can upgrade CO2 into valuable substances, but a multiplex monitoring system required for process control to maximize biomass productivity has not been well established. Herein, a 3D printed real-time optical density monitoring device (RTOMD) combined platform was presented. This platform enables precise kinetics analysis by maintaining high accuracy (over 95 %) under raucous outdoor conditions. Through RTOMD-based high-frequency measurements, it was observed that maximum biomass productivity of 4.497 g L-1 d-1 was reached, which greatly exceeds the requirements for a feasible microalgae process. We discovered that the CO2 fixation efficiency could be achieved to 70.75 %, indicating the potential of a bioconversion process to realize a carbon-neutral society. Consequently, the RTOMD system can contribute to promoting microalgae cultivation as an attractive carbon mitigation technology based on an improved understanding of the photosynthetic CO2 fixation kinetics.

A Rapid and High-Throughput Assay for Light Scattering of SARS-CoV-2 Virion-Sized Particulates via Microfluidic Spray Device Reveals the Protection Performance of Face Masks against Virus Infection.

To prevent interhuman transmission of viruses, new mask types─claiming improved filtration─require careful performance characterization. Here, a microfluidic spray device that can effectively simulate droplets emitted during coughing or sneezing was developed to spray droplets containing gold nanoparticles (AuNPs) that mimic SARS-CoV-2 to overcome the shortcomings associated with using biosamples. The light scattered by the AuNPs passing through the mask is successfully analyzed by using an automated scattering light mapping system within a duration of 2 min, thereby enabling high-throughput analysis of the filtering efficiency of various types of commercial masks. The differences in efficiency in terms of same mask type from different manufacturers, double masking, and prolonged usage, which are challenging to analyze with conventional testing systems, can also be assessed. AuNP-mediated mask performance evaluation enables the rapid determination of mask efficiency according to particle size and can contribute to the rapid response to counter new emerging infectious biohazards.

Multifaceted strategies for economic production of microalgae Haematococcus pluvialis-derived astaxanthin via direct conversion of CO2.

Owing to its strong antioxidant properties, astaxanthin has a high market price in the nutraceutical and pharmaceutical fields, and its demand is increasing. Furthermore, with an increase in the demand for green technology, astaxanthin production through direct CO2 conversion using the autotrophic green microalga Haematococcus pluvialis as a bio-platform has received much attention. Large-scale outdoor cultivation of H. pluvialis using waste CO2 sources and sunlight can secure sustainability and improve economic efficiency. However, low strain performance, reduced light utilization because of increased cell density, and inefficient transfer of gaseous CO2 into liquid culture broth hinder its large-scale commercialization of astaxanthin. Herein, we presented a multifaceted strategy, including the development of high-efficiency strains, a culture system for astaxanthin accumulation, and astaxanthin extraction from biomass, for economically producing astaxanthin from H. pluvialis through direct CO2 conversion. Future perspectives were presented by comparing and analyzing various previous studies conducted using the latest technology.

Concurrent enhancement of CO2 fixation and productivities of omega-3 fatty acids and astaxanthin in Haematococcus pluvialis culture via calcium-mediated homeoviscous adaptation and biomineralization.

Haematococcus pluvialis has attracted interest as a bio-platform for producing omega-3 fatty acids (ω-3 FA) and astaxanthin that have a great potential as anti-inflammatory drugs. This study aimed to concurrently enhance the CO2 fixation and the productivities of ω-3 FA and astaxanthin, which have been difficult to achieve because of the dissimilar culture methods for each goal, via calcium-mediated homeoviscous adaptation and biomineralization. As a result of 3 mM of Ca2+ addition, ω-3 FA content was improved by 31% due to Ca2+-induced homeoviscous adaptation. Biomineralization was promoted by the extracellular carbonic anhydrase, which resulted in 46.3% improvement in CO2 fixation. CaCO3 from the biomineralization was beneficially re-used in the H. pluvialis culture and triggered 178- and 522-fold increased biomass productivity and astaxanthin content, respectively, thanks to its anisotropic nature. The Ca2+-based productivity enhancement strategy was applied to large-scale culture which resulted improvement in overall bioprocess performance.

Robust cyst germination induction in Haematococcus pluvialis to enhance astaxanthin productivity in a semi-continuous outdoor culture system using power plant flue gas.

The microalgae Haematococcus pluvialis biologically converts CO2 into natural astaxanthin that possesses a strong antioxidant activity; its low carbon footprint and economic viability have allowed it to garner great attention. However, low efficiency of the cultivation process is a major hurdle in its commercial production. Here, additional nitrogen was provided to fully grown cells to induce efficient cyst germination, thereby rapidly increasing the number of cells containing astaxanthin. After germination, these cells were rapidly converted from zooids to cysts by nitrogen depletion, while maintaining a steady astaxanthin content of 5.5%. Consequently, a 2.1-fold increase in astaxanthin productivity in comparison with that in a batch culture was achieved. Moreover, the germination-based semi-continuous process yielded 2.6 times higher astaxanthin productivity in a large-scale culture using power plant flue gas and a polymeric photobioreactor. This study provides a promising cultivation strategy for the commercial mass production of natural astaxanthin.

A green decontamination technology through selective biomineralization of algicidal microorganisms for enhanced astaxanthin production from Haematococcus pluvialis at commercial scale.

Currently, there is a lack of an efficient, environmentally-benign and sustainable industrial decontamination strategy to steadily achieve improved astaxanthin production from Haematococcus pluvialis under large-scale outdoor conditions. Here, this study demonstrates for the first time that a CaCO3 biomineralization-based decontamination strategy (CBDS) is highly efficient in selectively eliminating algicidal microorganisms, such as bacteria and fungi, during large-scale H. pluvialis cultivation under autotrophic and mixotrophic conditions, thereby augmenting the astaxanthin productivity. Under outdoor AT and MT conditions, the average astaxanthin productivity of H. pluvialis using CBDS in a closed photobioreactor system was substantially increased by 14.85- (1.19 mg L-1 d-1) and 13.65-fold (2.43 mg L-1 d-1), respectively, compared to the contaminated H. pluvialis cultures. Given the exponentially increasing demand of astaxanthin, a natural anti-viral, anti-inflammatory, and antioxidant drug, CBDS will be a technology of interest in H. pluvialis-based commercial astaxanthin production which has been hindered by the serious biological contaminations.

Improved CO2-derived polyhydroxybutyrate (PHB) production by engineering fast-growing cyanobacterium Synechococcus elongatus UTEX 2973 for potential utilization of flue gas.

Industrial application of cyanobacterial poly-ß-hydroxybutyrate (PHB) production from CO2 is currently challenged by slow growth rate and low photoautotrophic PHB productivity of existing cyanobacteria species. Herein, a novel PHB-producing cyanobacterial strain was developed by harnessing fast-growing cyanobacteria Synechococcus elongatus UTEX 2973 with introduction of heterologous phaCAB genes. Under photoautotrophic condition, the engineered strain produced 420 mg L-1 (16.7% of dry cell weight) with the highest specific productivity of 75.2 mg L-1 d-1. When compared with a native PHB producer Synechocystis PCC 6803 under nitrogen deprivation, the engineered strain exhibited 2.4-fold higher PHB productivity. The performance of the engineered strain was further demonstrated in large scale cultivation using photobioreactor and outdoor cultivation employing industrial flue gas as the sole carbon source. This study can provide a promising solution to address petroleum-based plastic waste and contribute to CO2 mitigation.

Sedimentation rate-based screening of oleaginous microalgae for utilization as a direct combustion fuel.

The co-combustion of microalgae biomass with coal has the potential to significantly reduce CO2 emissions by eliminating expensive and carbon-emitting downstream processes. In this study, the utilization of microalgal biomass as a direct combustion fuel in co-firing industries and the screening of potential oleaginous strains of high calorific value was investigated. High-lipid accumulating mutants were selected from mutant mixtures based on cell density using differential sedimentation rates. Of the mutant strains obtained in the top phase of the separation medium, 72% showed a higher lipid content than the wild-type strain. One mutant strain exhibited a 57.3% enhanced lipid content and a 9.3% lower heating value (LHV), both indicators of direct combustion fuel performance, compared to the wild-type strain. Our findings indicate that sedimentation rate-based strain selection allows for the easy and rapid screening of high-lipid content algal strains for the use of microalgae as direct combustion fuels.

Screening of oleaginous algal strains from Chlamydomonas reinhardtii mutant libraries via density gradient centrifugation.

Microalgae have a high potential to be utilized as feedstock for biofuels because they have high growth rates and do not compromise food production. Commercialized algae-based biofuel production relies on the development of strains with high lipid content. Based on the relatively low density of lipids compared to other cellular components, density gradient centrifugation was used to isolate high lipid content algal strains from Chlamydomonas reinhardtii mutant libraries. The correlation between cell density and lipid content was confirmed by analysis of Nile red fluorescence intensity, total lipids, and total fatty acid methyl ester content. A strain isolated by this screening method had 50% higher lipid content and 7% lower cell density than the parent wild-type strain. Consequently, we demonstrated that screening of algal strains with low cell density via continuous density gradient centrifugation allows simple, rapid, and inexpensive screening for high lipid content strains.

Enhanced biomass and lipid production of Neochloris oleoabundans under high light conditions by anisotropic nature of light-splitting CaCO3 crystal.

The aim of this work was to study the anisotropic effect of crystalline CaCO3 nanoparticles (CN)-driven multiple refraction/scattering from the CN-coated agglomerated cells on the rate of photosynthesis and the product yield under high light conditions in the freshwater microalgae Neochloris oleoabundans. The CN-coating via biomineralization significantly improved the biomass and lipid production of N. oleoabundans during second stage of autotrophic induction by sustaining relatively high rate of photosynthesis at high irradiance using the multiple-splitting effect of the anisotropic polymorphism. The CN were successfully produced, adsorbed and grown on the external cells under conditions of mild alkalinity (pH 7.5-8.0), mild CaCl2 concentration (0.05 M) and under nitrogen starvation with strong light (400 µE m-2 s-1). Consequently, lipid content and productivity of N. oleoabundans cells cultured with 0.05 M CaCl2 increased by 18.4% and 31.5%, respectively, compared to the cells cultured with 0.05 M CaCl2 and acetazolamide to inhibit calcification.

Performance and potential appraisal of various microalgae as direct combustion fuel.

Direct combustion of biomass is considered the most effective and simple means to contribute to CO2 reduction. In this context, the life-cycle potential of microalgal solid fuel, which has been overlooked so far, was comprehensively scrutinized ranging from cultivation to direct combustion. According to the quantitative data, using the raw fuel was confirmed to offer great benefits over the conventional lipid-targeted microalgal fuel systems through exploiting all of the biomass' energy potential, thereby being able to significantly increase the energy yield from biomass. The solid fuel is shown to exhibit diverse positive aspects owing to its remarkable calorific value, productivity and CO2 fixation ability. The combustion test reveals coal-microalgae co-combustion brings beneficial consequences on combustibility and environmental impacts with no notable thermal efficiency drop. This holistic appraisal shows microalgae patently possess high potential as a direct combustion fuel, even outperforming that of extensively used woody fuels.

Two-Dimensional Microfluidic System for the Simultaneous Quantitative Analysis of Phototactic/Chemotactic Responses of Microalgae.

Microalgae have been spotlighted as a renewable energy source to produce biofuels from CO2 by photosynthesis. However, their innate inefficiency of CO2 conversion using light energy has been a challenge to the commercialization of algae-based biofuel production. Photosynthetic organisms have evolved behavioral responses, including phototaxis and chemotaxis, to find optimal conditions for capturing light energy and inorganic carbon (Ci) sources for photosynthesis. In this context, investigation of phototaxis and chemotaxis to HCO3-, the predominant form of Ci in neutral aqueous solutions, is necessary to understand the physiological role of tactic responses in photosynthesis. In this study, a two-dimensional microfluidic system enabled efficient analysis of phototactic and chemotactic responses by investigation of cell distribution in the outlet chambers. From statistical analysis (skewness and kurtosis) of tactic responses of different algal strains to external stimuli, the preferred concentrations of HCO3- for Chlamydomonas reinhardtii CC125 (wild type), PTS42 (random insertional mutant of C. reinhardtii, high photosynthetic activity), and CC2702 ( cia5 mutant of C. reinhardtii, unable to acclimate to low CO2 concentration) were determined to be 27.22, 43.23, and 36.95 mM, respectively. From the analysis of tactic responses of wild type and 14 mutant strains, it was found that the photosystem II (PSII) operating efficiency and CO2 fixation rate were strongly correlated with the phototactic ( R2 = 0.931) and chemotactic response ( R2 = 0.857), respectively. Finally, this system can be applied to high-throughput screening strategies for the rapid isolation of high photosynthetically productive microalgal strains based on their tactic responses.

Rapid selection of astaxanthin-hyperproducing Haematococcus mutant via azide-based colorimetric assay combined with oil-based astaxanthin extraction.

The aim of this work was to develop a new approach for simple and high-throughput selection of astaxanthin-hyperproducing Haematococcus mutants through a sequential combination method of azide-based colorimetric assessment and oil-based astaxanthin quantification. Randomly mutagenized cells were spotted on solid culture medium containing 50 µM of sodium azide to accelerate the biosynthesis of astaxanthin. After 3 days, highly-induced mutants were preliminarily isolated by visual inspection and their astaxanthin accumulations were rapidly quantified by soybean oil-based extraction method. On the whole, the selected mutants showed reduced vegetative growth rates but eventually exhibited higher astaxanthin productions than the parental strain owing to their improved inductive growths. Among them, M13 showed 174.7 ±â€¯5.69 mg L-1 of the highest astaxanthin production, which is 1.59-times higher than that of wild-type. This wide-scope screening method expedites both upstream and downstream astaxanthin quantification, making it a useful tool for isolating microalgae with high astaxanthin production.

Multilateral approach on enhancing economic viability of lipid production from microalgae: A review.

Microalgae have been rising as a feedstock for biofuel in response to the energy crisis. Due to a high lipid content, composed of fatty acids favorable for the biodiesel production, microalgae are still being investigated as an alternative to biodiesel. Environmental factors and process conditions can alternate the quality and the quantity of lipid produced by microalgae, which can be critical for the overall production of biodiesel. To maximize both the lipid content and the biomass productivity, it is necessary to start with robust algal strains and optimal physio-chemical properties of the culture environment in combination with a novel culture system. These accumulative approaches for cost reduction can take algal process one step closer in achieving the economic feasibility.

Magnetophoretic sorting of microdroplets with different microalgal cell densities for rapid isolation of fast growing strains.

Microalgae - unicellular photosynthetic organisms - have received increasing attention for their ability to biologically convert CO2 into valuable products. The commercial use of microalgae requires screening strains to improve the biomass productivity to achieve a high-throughput. Here, we developed a microfluidic method that uses a magnetic field to separate the microdroplets containing different concentrations of microalgal cells. The separation efficiency is maximized using the following parameters that influence the amount of lateral displacement of the microdroplets: magnetic nanoparticle concentration, flow rate of droplets, x- and y-axis location of the magnet, and diameter of the droplets. Consequently, 91.90% of empty, 87.12% of low-, and 90.66% of high-density droplets could be separated into different outlets through simple manipulation of the magnetic field in the microfluidic device. These results indicate that cell density-based separation of microdroplets using a magnetic force can provide a promising platform to isolate microalgal species with a high growth performance.

Quantitative analysis of the chemotaxis of a green alga, Chlamydomonas reinhardtii, to bicarbonate using diffusion-based microfluidic device.

There is a growing interest in the photosynthetic carbon fixation by microalgae for the production of valuable products from carbon dioxide (CO2). Microalgae are capable of transporting bicarbonate (HCO3 (-)), the most abundant form of inorganic carbon species in the water, as a source of CO2 for photosynthesis. Despite the importance of HCO3 (-) as the carbon source, little is known about the chemotactic response of microalgae to HCO3 (-). Here, we showed the chemotaxis of a model alga, Chlamydomonas reinhardtii, towards HCO3 (-) using an agarose gel-based microfluidic device with a flow-free and stable chemical gradient during the entire assay period. The device was validated by analyzing the chemotactic responses of C. reinhardtii to the previously known chemoattractants (NH4Cl and CoCl2) and chemotactically neutral molecule (NaCl). We found that C. reinhardtii exhibited the strongest chemotactic response to bicarbonate at the concentration of 26 mM in a microfluidic device. The chemotactic response to bicarbonate showed a circadian rhythm with a peak during the dark period and a valley during the light period. We also observed the changes in the chemotaxis to bicarbonate by an inhibitor of bicarbonate transporters and a mutation in CIA5, a transcriptional regulator of carbon concentrating mechanism, indicating the relationship between chemotaxis to bicarbonate and inorganic carbon metabolism in C. reinhardtii. To the best of our knowledge, this is the first report of the chemotaxis of C. reinhardtii towards HCO3 (-), which contributes to the understanding of the physiological role of the chemotaxis to bicarbonate and its relevance to inorganic carbon utilization.

Microfluidic high-throughput selection of microalgal strains with superior photosynthetic productivity using competitive phototaxis.

Microalgae possess great potential as a source of sustainable energy, but the intrinsic inefficiency of photosynthesis is a major challenge to realize this potential. Photosynthetic organisms evolved phototaxis to find optimal light condition for photosynthesis. Here we report a microfluidic screening using competitive phototaxis of the model alga, Chlamydomonas reinhardtii, for rapid isolation of strains with improved photosynthetic efficiencies. We demonstrated strong relationship between phototaxis and photosynthetic efficiency by quantitative analysis of phototactic response at the single-cell level using a microfluidic system. Based on this positive relationship, we enriched the strains with improved photosynthetic efficiency by isolating cells showing fast phototactic responses from a mixture of 10,000 mutants, thereby greatly improving selection efficiency over 8 fold. Among 147 strains isolated after screening, 94.6% showed improved photoautotrophic growth over the parental strain. Two mutants showed much improved performances with up to 1.9- and 8.1-fold increases in photoautotrophic cell growth and lipid production, respectively, a substantial improvement over previous approaches. We identified candidate genes that might be responsible for fast phototactic response and improved photosynthesis, which can be useful target for further strain engineering. Our approach provides a powerful screening tool for rapid improvement of microalgal strains to enhance photosynthetic productivity.

Microdroplet photobioreactor for the photoautotrophic culture of microalgal cells.

Microalgae, unicellular photoautotrophic microorganisms, have attracted great attention for the production of biofuel and high-value products, but the commercial use of microalgae has been limited by low photosynthetic productivity. To overcome this limitation, it is required to develop an efficient platform for the rapid evaluation of photoautotrophic growth performance and productivity of microalgal strains. Here we describe a droplet-based photobioreactor for high-throughput analysis of the photoautotrophic growth of microalgal cells. By integrating micropillar arrays and adjusting the height of the microchamber, we could accurately monitor the growth kinetics of microalgae in an immobilized microdroplet and improve the transfer rate of CO2 into the microdroplet photobioreactor with an increased contact area between the microdroplet and PDMS surface. The improvement of CO2 transfer into the microdroplet was also confirmed by improved microalgal cell growth and a decrease in pH measured using colorimetric and fluorescence-based assays. The photoautotrophic growth kinetics of Chlorella vulgaris were measured under different CO2 concentrations (ambient, 1%, 2.5%, 5% and 7.5%) and light intensity (35, 55, 100, 150, and 200 µmol photons per m(2) per s) conditions, which are key factors for photoautotrophic growth. Chlorella vulgaris in a microdroplet showed better cell growth performance compared to a flask culture due to the reduced shading effects and improved mass transfer. Finally, we could evaluate the photoautotrophic growth performance of four microalgal strains (Chlorella vulgaris, Chlorella protothecoides, Chlorella sorokiniana and Neochloris oleoabundans) for 120 hours. These results demonstrate that our microdroplet system can be used as an efficient photobioreactor for the rapid evaluation of the photoautotrophic growth of microalgal strains under various conditions.