Genome editing is crucial for genetic engineering of organisms for improved traits, particularly in microalgae due to the urgent necessity for the next generation biofuel production. The most ...advanced CRISPR/Cas9 system is simple, efficient and accurate in some organisms; however, it has proven extremely difficult in microalgae including the model alga Chlamydomonas. We solved this problem by delivering Cas9 ribonucleoproteins (RNPs) comprising the Cas9 protein and sgRNAs to avoid cytotoxicity and off-targeting associated with vector-driven expression of Cas9. We obtained CRISPR/Cas9-induced mutations at three loci including MAA7, CpSRP43 and ChlM, and targeted mutagenic efficiency was improved up to 100 fold compared to the first report of transgenic Cas9-induced mutagenesis. Interestingly, we found that unrelated vectors used for the selection purpose were predominantly integrated at the Cas9 cut site, indicative of NHEJ-mediated knock-in events. As expected with Cas9 RNPs, no off-targeting was found in one of the mutagenic screens. In conclusion, we improved the knockout efficiency by using Cas9 RNPs, which opens great opportunities not only for biological research but also industrial applications in Chlamydomonas and other microalgae. Findings of the NHEJ-mediated knock-in events will allow applications of the CRISPR/Cas9 system in microalgae, including "safe harboring" techniques shown in other organisms.
•Orange peel extract was used for microalgae cultivation.•This extract contains nutrients that support mixotrophic cultivation of microalgae.•Cells cultured in OPE produced significantly increased ...biomass and FAME.
Mass cultivation of microalgae is necessary to achieve economically feasible production of microalgal biodiesel, but the high cost of nutrients is a major limitation. In this study, orange peel extract (OPE) was used as an inorganic and organic nutrient source for the cultivation of Chlorella vulgaris OW-01. Chemical composition analysis of the OPE indicated that it contains sufficient nutrients for mixotrophic cultivation of C. vulgaris OW-01. Analysis of biomass and FAME production showed that microalgae grown in OPE medium produced 3.4-times more biomass and 4.5-times more fatty acid methyl esters (FAMEs) than cells cultured in glucose-supplemented BG 11 medium (BG-G). These results suggest that growth of microalgae in an OPE-supplemented medium increases lipid production and that OPE has potential for use in the mass cultivation of microalgae.
Microalgae have great potential for the production of biofuels due to the ability of the organism to accumulate large quantities of storage lipids under stress conditions. Mitogen activated protein ...kinase (MAPK) signaling cascades are widely recognized for their role in stress response signal transduction in eukaryotes. To assess the correlation between MAPK activation and lipid productivity, Chlamydomonas reinhardtii was studied under various concentrations of NaCl. The results demonstrated that C. reinhardtii exhibits elevated levels of extracellular-signal regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) activities after undergoing osmotic stress, as well as an increase in cellular lipid content. To establish a more direct causal link between both kinases and lipid productivity, C. reinhardtii was subjected to biochemically induced regulation of ERK and JNK pathways. Activating the MEK-ERK pathway via C6 ceramide treatment increased ERK activation and lipid production simultaneously, while PD98059 mediated inhibition of the pathway yielded opposite results. Interestingly, suppression of the JNK pathway with SP600125 resulted in a substantial decrease in cell viability under osmotic stress. These results suggest that ERK and JNK MAP kinases have important roles in microalgal lipid accumulation and cell growth under osmotic stress, respectively.
Due to the 4th industrial revolution and the strength of the 5 th Generation (5G) era, the Internet of Things (IoT) industry is growing significantly. As a result, the number of IoT devices in ...various industries, such as smart cars, smart homes, and smart healthcare, and the importance of security for these devices are increasing. This study proposes a design method for a secure cryptographic system on a chip (SecSoC) that can be used in the IoT industry and presents the results of the performance and security evaluations of the implemented chipset. The experimental results demonstrated that the SecSoC is a low-power high-performance cryptographic chip that is safe from external attacks. Compared to conventional smart card integrated circuits, the proposed design includes intrusion detection circuits that can respond to external attacks. At the same time, it supports a physical unclonable function for hiding secret data and cryptographic logic for maintaining integrity and confidentiality. The SecSoC ensured a fast transfer rate up to 110 Mbps and consumed only 95.8 mW when operating at maximum frequency.
Acyl-homoserine lactone–mediated quorum sensing (QS) regulates diverse activities in many species of Proteobacteria . QS-controlled genes commonly code for production of secreted or excreted public ...goods. The acyl-homoserine lactones are synthesized by members of the LuxI signal synthase family and are detected by cognate members of the LuxR family of transcriptional regulators. QS affords a means of population density-dependent gene regulation. Control of public goods via QS provides a fitness benefit. Another potential role for QS is to anticipate overcrowding. As population density increases and stationary phase approaches, QS might induce functions important for existence in stationary phase. Here we provide evidence that in three related species of the genus Burkholderia QS allows individuals to anticipate and survive stationary-phase stress. Survival requires QS-dependent activation of cellular enzymes required for production of excreted oxalate, which serves to counteract ammonia-mediated alkaline toxicity during stationary phase. Our findings provide an example of QS serving as a means to anticipate stationary phase or life at the carrying capacity of a population by activating the expression of cytoplasmic enzymes, altering cellular metabolism, and producing a shared resource or public good, oxalate.
Scenedesmus obliquus ABC-009 is a microalgal strain that accumulates large amounts of lutein, particularly when subjected to growth-limiting conditions. Here, the performance of this strain was ...evaluated for the simultaneous production of lutein and biofuels under three different modes of cultivation - photoautotrophic mode using BG-11 medium with air or 2% CO 2 and heterotrophic mode using YM medium. While it was found that the highest fatty acid methyl ester (FAME) level and lutein content per biomass (%) were achieved in BG-11 medium with CO 2 and air, respectively, heterotrophic cultivation resulted in much higher biomass productivity. While the cell concentrations of the cultures grown under BG-11 and CO 2 were largely similar to those grown in YM medium, the disparity in the biomass yield was largely attributed to the larger cell volume in heterotrophically cultivated cells. Post-cultivation light treatment was found to further enhance the biomass productivity in all three cases and lutein content in heterotrophic conditions. Consequently, the maximum biomass (757.14 ± 20.20 mg/l/d), FAME (92.78 ± 0.08 mg/l/d), and lutein (1.006 ± 0.23 mg/l/d) productivities were obtained under heterotrophic cultivation. Next, large-scale lutein production using microalgae was demonstrated using a 1-ton open raceway pond cultivation system and a low-cost fertilizer (Eco-Sol). The overall biomass yields were similar in both media, while slightly higher lutein content was obtained using the fertilizer owing to the higher nitrogen content.
In this study, we used the Monte Carlo N-Particle program to simulate the gamma-ray spectra obtained from plastic scintillators holes filled with bismuth nanoparticles. We confirmed that the ...incorporation of bismuth nanoparticles into a plastic scintillator enhances its performance for gamma-ray spectroscopy using the subtraction method. The subtracted energy spectra obtained from the bismuth-nanoparticle-incorporated and the original plastic scintillator exhibit a distinct energy peak that does not appear in the corresponding original spectra. We varied the diameter and depth of the bismuth-filled holes to determine the optimal hole design for gamma-ray spectroscopy using the subtraction method. We evaluated the energy resolutions of the energy peaks in the gamma-ray spectra to estimate the effects of the bismuth nanoparticles and determine their optimum volume in the plastic scintillator. In addition, we calculated the peak-to-total ratio of the energy spectrum to evaluate the energy measuring limit of the bismuth nanoparticle-containing plastic scintillator using the subtraction method.
We developed a miniaturized multi-dimensional radiation sensor system consisting of an inorganic scintillator array and plastic optical fibers. This system can be applied to remotely obtain the ...radioactivity distribution and identify the radionuclides in radioactive waste by utilizing a scanning method. Variation in scintillation light was measured in two-dimensional regions of interest and then converted into radioactivity distribution images. Outliers present in the images were removed by using a digital filter to make the hot spot location more accurate and cubic interpolation was applied to make the images smoother and clearer. Next, gamma-ray spectroscopy was performed to identify the radionuclides, and three-dimensional volume scanning was also performed to effectively find the hot spot using the proposed array sensor.
In this study, we fabricated a liquid light-guide-based radiation sensor with a LaBr3:Ce scintillator for remote gamma-ray spectroscopy. We acquired the energy spectra of Cs-137 and Co-60 using the ...proposed sensor, estimated the energy resolutions of the full energy peaks, and compared the scintillation light output variations. The major peaks of the radionuclides were observed in each result, and the estimated energy resolutions were similar to that of a general NaI(Tl) scintillation detector without a liquid light guide. Moreover, we showed the relationships of energy resolution and analog-to-digital channel regarding the number of photoelectrons produced and confirmed the effects of light guide length on remote gamma-ray spectroscopy. The proposed sensor is expected to be utilized to perform remote gamma-ray spectroscopy for distances of 3 m or more and would find application in many fields of nuclear facilities and industry.
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