Improved Productivity of Astaxanthin from Photosensitive Haematococcus pluvialis Using Phototaxis Technology.

Haematococcus pluvialis is a microalgae actively studied for the production of natural astaxanthin, which is a powerful antioxidant for human application. However, it is economically disadvantageous for commercialization owing to the low productivity of astaxanthin. This study reports an effective screening strategy using the negative phototaxis of the H. pluvialis to attain the mutants having high astaxanthin production. A polydimethylsiloxane (PDMS)-based microfluidic device irradiated with a specific light was developed to efficiently figure out the phototactic response of H. pluvialis. The partial photosynthesis deficient (PP) mutant (negative control) showed a 0.78-fold decreased cellular response to blue light compared to the wild type, demonstrating the positive relationship between the photosynthetic efficiency and the phototaxis. Based on this relationship, the Haematococcus mutants showing photosensitivity to blue light were selected from the 10,000 random mutant libraries. The M1 strain attained from the phototaxis-based screening showed 1.17-fold improved growth rate and 1.26-fold increases in astaxanthin production (55.12 ± 4.12 mg g-1) in the 100 L photo-bioreactor compared to the wild type. This study provides an effective selection tool for industrial application of the H. pluvialis with improved astaxanthin productivity.

Pregnancy and neonatal outcomes after periconceptional exposure to isotretinoin in Koreans.

OBJECTIVE: Isotretinoin should not be used during pregnancy because of the risk of birth defects. Most pregnant women exposed to isotretinoin choose voluntary pregnancy termination due to concerns about birth defects. However, birth outcome data supporting the termination of pregnancy are lacking. This study aimed to evaluate pregnancy and neonatal outcomes after periconception exposure to isotretinoin. METHODS: This was a prospective cohort study. We evaluated pregnancy and neonatal outcomes after exposure to isotretinoin in 151 pregnant women. Among 1,026 callers at the Korean Teratology Information Service from 2001 to 2017 exposed to isotretinoin during the periconception period, 151 pregnant women who received counseling on teratogenic risk after visiting the clinic were included. RESULTS: Among the 151 participants who visited the clinic, only 42 were evaluated using ultrasonography until approximately 20 weeks of gestation. Ultimately, 23 patients were included in the study. The average gestation period during the last exposure to the drug was 2 weeks, and the average daily exposure dose was 12 mg. There were two cases of major birth defects in the exposure group. Spontaneous abortion rates were 17.7% and 8.7% in the exposure and nonexposure groups, respectively (P=0.035). There was no significant difference between the exposure and non-exposure groups in terms of pregnancy and neonatal outcomes. CONCLUSION: There was no significant difference in pregnancy and neonatal outcomes, including birth defects, between the exposure and non-exposure groups. Further studies with larger sample sizes are required to validate our findings.

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.

The rates of major malformations after gestational exposure to isotretinoin: a systematic review and meta-analysis.

OBJECTIVE: Isotretinoin is among the most notorious human teratogens, documented originally as causing up to 30% of malformations. This systematic review and meta-analysis aimed to evaluate the rates of major malformation (MM) among isotretinoin-exposed pregnant women over the years through a systematic review and meta-analysis. METHODS: Eligible studies were searched and identified using various databases. Single-arm meta-analysis and meta-analysis of odd ratios among controlled studies were performed using Review Manager version 5.3. RESULTS: Ten eligible studies that combined 2,783 isotretinoin-exposed women were included in our study. The rate of MM weighted for the sample size was 15%. Three studies that included an unexposed comparison group were eligible for the meta-analysis. The pooled odds ratio of MM for isotretinoin-exposed women was 3.76. After 2006, the pooled odds ratio of MM for isotretinoin exposure was significantly lower at 1.04. CONCLUSION: The current rate of MM in isotretinoin-exposed women was substantially lower after 2006.

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.

Association of Fatty Acid Ethyl Esters in Meconium of Neonates with Growth Deficits at Birth: a Prospective, Single-Centre Cohort Study.

BACKGROUND: In this prospective cohort study, we investigated the association between fatty acid ethyl esters (FAEEs) in meconium as biomarkers of prenatal ethanol exposure and growth deficits, as birth outcomes, that constitute several of the key cardinal features of fetal alcohol syndrome. METHODS: A total of 157 meconium samples were collected from enrolled infants within 24 hours of birth, and nine FAEEs were quantified using liquid chromatography/tandem mass spectrometry. The relationships between cumulative concentrations of nine species of FAEEs in meconium and birth parameters of growth (age-sex-specific centiles of head circumference [HC], weight, and length) and respective and combined birth outcomes of growth deficits (HC ≤ 10th centile, weight ≤ 10th centile, and length ≤ 10th centile) were determined. RESULTS: Multivariate logistic regression analysis demonstrated that higher cumulative concentrations of meconium FAEEs correlated with elevated risks for HC and length, both, 10th percentile or less (adjusted odds ratio [aOR], 2.94; 95% confidence interval [CI], 1.12-7.74; P = 0.029) and HC and weight and length, all of them, 10th percentile or less (aOR, 3.27; 95% CI, 1.12-9.59; P = 0.031). CONCLUSION: The elevated cumulative FAEEs in meconium were associated with combined growth deficits at birth, specifically HC and length, both, 10th percentile or less, which might be correlated with detrimental alcohol effects on fetal brain and bone development, suggesting a plausible alcohol-specific pattern of intrauterine growth restriction.

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.

Smoking Exposure in Early Pregnancy and Adverse Pregnancy Outcomes: Usefulness of Urinary Tobacco-Specific Nitrosamine Metabolite 4-(Methylnitrosamino)-1-(3-Pyridyl)-1-Butanol Levels.

OBJECTIVES: The aim was to investigate the effect of -maternal smoking exposure assessed by urinary tobacco-specific nitrosamine metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-a1-butanol (NNAL) with adverse pregnancy outcomes. METHODS: A total of 251 pregnant women were recruited. Urinary cotinine and NNAL were measured. Participants' sociodemographics were obtained by questionnaire and pregnancy outcomes were collected by charts review after delivery. RESULTS: The prevalence of smoking was 8.4% (21 of 249), 1.2% (3 of 241), and 3.7% (9 of 241) in pregnant women according to questionnaire, cotinine, and NNAL, respectively. As compared with questionnaire positivity and cotinine levels, women with positive NNAL were independent determinants for spontaneous abortion (adjusted OR 12.357, 95% CI 2.053-74.368), preterm birth (adjusted OR 22.239, 95% CI 3.737-132.357), and small for gestational age (adjusted OR 6.915, 95% CI 1.385-34.524). CONCLUSIONS: Urinary NNAL might be a useful biomarker in detection of maternal smoking status in association with adverse pregnancy outcomes. Use of this marker in preconception and pregnancy counselling before planning pregnancy may allow prevention of several adverse pregnancy outcomes.

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.

Development of an X-Shape airlift photobioreactor for increasing algal biomass and biodiesel production.

The aim of this work was to develop a high efficient photobioreactor for increasing biomass and lipid production in microalgae by assessment of the hydrodynamic properties and kLa which are important parameters for improving the algal cultivation efficiency. We designed three different photobioreactors (H-Shape, X-Shape and serial-column). Among them, X-Shape showed the highest hydrodynamic properties and kLa for algal cultivation. Thus, we evaluated the biomass and the lipid production in a 20L scale-up X-Shape photobioreactor. The biomass and lipid production from X-Shape photobioreactor are 1.359±0.007gL-1 and 117.624±3.522mgL-1, respectively; which are 30.05% and 23.49% higher than those from the control photobioreactor. Finally, we observed the lipid from X-Shape had high MUFAs, CN and low IV, which is suitable for high quality of biodiesel, suggesting that it can be practicably utilized for mass production of algal biofuel.

Improvement of Squalene Production from CO2 in Synechococcus elongatus PCC 7942 by Metabolic Engineering and Scalable Production in a Photobioreactor.

The push-and-pull strategy for metabolic engineering was successfully demonstrated in Synechococcus elongatus PCC 7942, a model photosynthetic bacterium, to produce squalene from CO2. Squalene synthase (SQS) was fused to either a key enzyme (farnesyl diphosphate synthase) of the methylerythritol phosphate pathway or the ß-subunit of phycocyanin (CpcB1). Engineered cyanobacteria with expression of a fusion CpcB1-SQS protein showed a squalene production level (7.16 ± 0.05 mg/L/OD730) that was increased by 1.8-fold compared to that of the control strain expressing SQS alone. To increase squalene production further, the gene dosage for CpcB1·SQS protein expression was increased and the fusion protein was expressed under a strong promoter, yielding 11.98 ± 0.49 mg/L/OD730 of squalene, representing a 3.1-fold increase compared to the control. Subsequently, the best squalene producer was cultivated in a scalable photobioreactor (6 L) with light optimization, which produced 7.08 ± 0.5 mg/L/OD730 squalene (equivalent to 79.2 mg per g dry cell weight). Further optimization for photobioprocessing and strain development will promote the construction of a solar-to-chemical platform.

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.

Multiplex microfluidic system integrating sequential operations of microalgal lipid production.

The unit cost for the production of algal biofuel needs to be reduced in order to be a substitute for fossil fuel. To achieve this goal, the development of a novel system is needed for a rapid screening of numerous microalgal species to isolate superior strains with the highest lipid productivity. Here, we developed a PDMS-based multiplex microfluidic system with eight chambers and micropillar arrays to expedite multiple steps for lipid sample preparation from different microalgal strains. We could rapidly and efficiently perform sequential operations from cell culture to lipid extraction of eight different microalgal strains simultaneously on a single device without harvesting and purification steps, which are labor- and energy-intensive, by the simple injection of medium and solvent into the central inlet due to the integrated micropillar arrays connecting the chambers and central inlet. The lipid extraction efficiency using this system was comparable (94.5-102.6%) to the conventional Bligh-Dyer method. We investigated the cell growth and lipid productivity of different strains using the microfluidic device. We observed that each strain has a different lipid accumulation pattern according to stress conditions. These results demonstrate that our multiplex microfluidic approach can provide an efficient analytical tool for the rapid analysis of strain performances (e.g. cell growth and lipid productivities) and the determination of the optimal lipid induction condition for each strain.

Could a first-trimester blood phosphatidylethanol concentration ⩾4 nM be useful to identify women with moderate-to-heavy prenatal alcohol exposure who are at high risk of adverse pregnancy outcomes?

It is accepted that blood phosphatidylethanol (PEth) concentrations are reliable biomarkers of ethanol (alcohol) exposure. We therefore conducted a preliminary study to test the hypothesis that elevated blood PEth concentrations can help to identifying women with prenatal alcohol exposure who are at higher risk of adverse pregnancy outcomes. The study included 35 first-trimester pregnant women who self-reported alcohol ingestion and had PEth blood concentration ⩾4 nM at recruitment. As a control group, 233 first-trimester pregnant women who self-reported as being either abstainers or light alcohol drinkers and had blood PEth concentrations <4 nM, were also included. All participants were followed up until completion of their pregnancies. Women with prenatal alcohol exposure and PEth concentrations ⩾4 nM had a risk ratio of spontaneous abortions of 3.21 (95%CI 0.93-11.06; P=0.074). Because of the potential implications in the prenatal care of women reporting risky alcohol exposure, the preliminary results from the present study indicate the need for testing the hypothesis in a more definitive approach.

A Microreactor System for Cultivation of Haematococcus pluvialis and Astaxanthin Production.

Development of efficient culture and monitoring system for cell growth and production of useful materials is required for practical utilization of microalgae. In the present study, we developed a PDMS-based microreactor system for efficient, rapid culture of microalgae and monitoring of cell growth, carotenoid content under diverse culture conditions. Due to advantages of PDMS, we optimized culture conditions (light intensity, pH, nitrate depletion, carbon dioxide concentration) for improving growth rate and astaxanthin productivity in considerably less time compared to conventional culture methods using flask or well plate. In addition, we found that there was a strong linear correlation between fluorescence intensity of astaxanthin stained by Nile red and the astaxanthin content, which can be utilized as a high-throughput screening tool in microfluidic systems. In this study, the growth rate of vegetative Haematococcus pluvialis was improved by 60% in microfluidic chamber than in flask and astaxanthin was produced up to 362 mg/L under the optimal conditions (300 µmol photon/m2/s of light, 7% CO2 (v/v), and pH 7.0) using designed microfluidic devices. This result shows that microfluidic system can provide effective means to address development of microalgal strains including H. pluvialis and bioprocess.

A microscale approach for simple and rapid monitoring of cell growth and lipid accumulation in Neochloris oleoabundans.

Due to the increasing environmental problems caused by the use of fossil fuels, microalgae have been spotlighted as renewable resources to produce biomass and biofuels. Therefore, the investigation of the optimum culture conditions of microalgae in a short time is one of the important factors for improving growth and lipid productivity. Herein, we developed a PDMS-based high-throughput screening system to rapidly and easily determine the optimum conditions for high-density culture and lipid accumulation of Neochloris oleoabundans. Using the microreactor, we were able to find the optimal culture conditions of N. oleoabundans within 5 days by rapid and parallel monitoring growth and lipid induction under diverse conditions of light intensity, pH, CO2 and nitrate concentration. We found that the maximum growth rate (µ max = 2.13 day(-1)) achieved in the microreactor was 1.58-fold higher than that in a flask (µ max = 1.34 day(-1)) at the light intensity of 40 µmol photons m(-2) s(-1), 5 % CO2 (v/v), pH 7.5 and 7 mM nitrate. In addition, we observed that the accumulation of lipid in the microreactor was 1.5-fold faster than in a flask under optimum culture condition. These results show that the microscale approach has the great potential for improving growth and lipid productivity by high-throughput screening of diverse optimum conditions.

Prediction of fetal lung maturity using the lecithin/sphingomyelin (L/S) ratio analysis with a simplified sample preparation, using a commercial microtip-column combined with mass spectrometric analysis.

Fetal lung maturity is estimated using the lecithin/sphingomyelin ratio (L/S ratio) in amniotic fluid and it is commonly measured with thin-layer chromatography (TLC). The TLC method is time consuming and technically difficult; however, it is widely used because there is no alternative. We evaluated a novel method for measuring the L/S ratio, which involves a tip-column with a cation-exchange resin and mass spectrometry. Phospholipids in the amniotic fluid were extracted using methanol and chloroform. Choline-containing phospholipids such as lecithin and sphingomyelin were purified by passing them through the tip-column. LC-MS/MS and MALDI-TOF were used to directly analyze the purified samples. The L/S ratio by mass spectrometry was calculated from the sum peak intensity of the six lecithin, and that of sphingomyelin 34:1. In 20 samples, the L/S ratio determined with TLC was significantly correlated with that obtained by LC-MS/MS and MALDI-TOF. There was a 100% concordance between the L/S ratio by TLC and that by LC-MS/MS (kappa value=1.0). The concordance between the L/S ratio by TLC and that by MALDI-TOF was also 100% (kappa value=1.0). Our method provides a faster, simpler, and more reliable assessment of fetal lung maturity. The L/S ratio measured by LC-MS/MS and MALDI-TOF offers a compelling alternative method to traditional TLC.