Cost-effective production of bioplastic polyhydroxybutyrate via introducing heterogeneous constitutive promoter and elevating acetyl-Coenzyme A pool of rapidly growing cyanobacteria.

Bioplastic production using cyanobacteria can be an effective strategy to cope with environmental problems caused by using petroleum-based plastics. Synechococcus elongatus UTEX 2973 with heterogeneous phaCAB can produce bioplastic polyhydroxybutyrate (PHB) with a high CO2 uptake rate. For cost-effective production of PHB in S. elongatus UTEX 2973, phaCAB was expressed by the constitutive Pcpc560, resulting in the production of 226 mg/L of PHB by only photoautotrophic cultivation without the addition of inducer. Several culture conditions were applied to increase PHB productivity, and when acetate was supplied at a concentration of 1 g/L as an organic carbon source, productivity significantly increased resulting in 607.2 mg/L of PHB and additive cost reduction of more than 300 times was achieved compared to IPTG. Consequently, these results suggest the possibility of cyanobacteria as an agent that can economically produce PHB and as a solution to the problem of petroleum-based plastics.

Lateral flow immunoassay based on surface-enhanced Raman scattering using pH-induced phage-templated hierarchical plasmonic assembly for point-of-care diagnosis of infectious disease.

The outbreak of emerging infectious diseases gave rise to the demand for reliable point-of-care testing methods to diagnose and manage those diseases in early onset. However, the current on-site testing methods including lateral flow immunoassay (LFIA) suffer from the inaccurate diagnostic result due to the low sensitivity. Herein, we present the surface-enhanced Raman scattering-based lateral flow immunoassay (SERS-LFIA) by introducing phage-templated hierarchical plasmonic assembly (PHPA) nanoprobes to diagnose a contagious disease. The PHPA was fabricated using gold nanoparticles (AuNPs) assembled on bacteriophage MS2, where inter-particle gap sizes can be adjusted by pH-induced morphological alteration of MS2 coat proteins to provide the maximum SERS amplification efficiency via plasmon coupling. The plasmonic probes based on the PHPA produce strong and reproducible SERS signal that leads to sensitive and reliable diagnostic results in SERS-LFIA. The developed SERS-LFIA targeting severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) antibodies for a proof of concept had <100 pg/mL detection limits with high specificity in serum, proving it as an effective diagnostic device for the infectious diseases. Clinical validation using human serum samples further confirmed that the PHPA-based SERS-LFIA can distinguish the patients with COVID-19 from healthy controls with significant accuracy. These outcomes prove that the developed SERS-LFIA biosensor can be an alternative point-of-care testing (POCT) method against the emerging infectious diseases, in combination with the commercially available portable Raman devices.

Distinct plasma phosphorylated-tau proteins profiling for the differential diagnosis of mild cognitive impairment and Alzheimer's disease by plasmonic asymmetric nanobridge-based biosensor.

The differential diagnosis between mild cognitive impairment (MCI) and Alzheimer's disease (AD) has been highly demanded for its effectiveness in preventing and contributing to early diagnosis of AD. To this end, we developed a single plasmonic asymmetric nanobridge (PAN)-based biosensor to differentially diagnose MCI and AD by quantitative profiling of phosphorylated tau proteins (p-tau) in clinical plasma samples, which revealed a significant correlation with AD development and progression. The PAN was designed to have a conductive junction and asymmetric structure, which was unable to be synthesized by the traditional thermodynamical methods. For its unique morphological characteristics, PAN features high electromagnetic field enhancement, enabling the biosensor to achieve high sensitivity, with a limit of detection in the attomolar regime for quantitative analysis of p-tau. By introducing support vector machine (SVM)-based machine learning algorithm, the improved diagnostic system was achieved for prediction of healthy controls, MCI, and AD groups with an accuracy of 94.47 % by detecting various p-tau species levels in human plasma. Thus, our proposed PAN-based plasmonic biosensor has a powerful potential in clinical utility for predicting the onset of AD progression in the asymptomatic phase.

Bio-conjugated nanoarchitectonics with dual-labeled nanoparticles for a colorimetric and fluorescent dual-mode serological lateral flow immunoassay sensor in detection of SARS-CoV-2 in clinical samples.

Serological detection of antibodies for diagnosing infectious diseases has advantages in facile diagnostic procedures, thereby contributing to controlling the spread of the pathogen, such as in the recent SARS-CoV-2 pandemic. Lateral flow immunoassay (LFIA) is a representative serological antibody detection method suitable for on-site applications but suffers from low clinical accuracy. To achieve a simple and rapid serological screening as well as the sensitive quantification of antibodies against SARS-CoV-2, a colorimetric and fluorescent dual-mode serological LFIA sensor incorporating metal-enhanced fluorescence (MEF) was developed. For the strong fluorescence signal amplification, fluorophore Cy3 was immobilized onto gold nanoparticles (AuNPs) with size-controllable spacer polyethyleneglycol (PEG) to maintain an optimal distance to induce MEF. The sensor detects the target IgG with a concentration as low as 1 ng mL-1 within 8 minutes. The employment of the MEF into the dual-mode serological LFIA sensor shows a 1000-fold sensitivity improvement compared with that of colorimetric LFIAs. The proposed serological LFIA sensor was tested with 73 clinical samples, showing sensitivity, specificity, and accuracy of 95%, 100%, and 97%, respectively. In conclusion, the dual-mode serological LFIA has great potential for application in diagnosis and an epidemiological survey of vaccine efficacy and immunity status of individuals.

A strategy to maximize astaxanthin production from Haematococcus pluvialis in a cost-effective process by utilizing a PBR-LGP-PBR array (PLPA) hybrid system using light guide panel (LGP) and solar cells.

This study evaluated economic feasibility through production efficiency, return on investment (ROI) and payout time of a hybrid system using a photobioreactor (PBR)-light guide panel (LGP)-PBR array (PLPA) and solar cells developed for astaxanthin and ω-3 FA simultaneous production of Haematococcus pluvialis. The economic feasibility of the PLPA hybrid system (8 PBRs) and the PBR-PBR-PBR array (PPPA) system (8 PBRs) was evaluated for producing high-value products while effectively reducing CO2. Introducing a PLPA hybrid system has increased the amount of culture per area by 1.6 times. Also, the shading effect was effectively suppressed with an LGP placed between each PBR, increasing biomass and astaxanthin productivity by 3.39-fold and 4.79-fold, respectively compared to the untreated H. pluvialis cultures. In addition, ROI increased by 6.55 and 4.71 times, and the payout time was reduced by 1.34 and 1.37 times, respectively in 10 and 100-ton scale processes.

Precise profiling of exosomal biomarkers via programmable curved plasmonic nanoarchitecture-based biosensor for clinical diagnosis of Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disease of complex pathogenesis, with overt symptoms following disease progression. Early AD diagnosis is challenging due to the lack of robust biomarkers and limited patient access to diagnostics via neuroimaging and cerebrospinal fluid (CSF) tests. Exosomes present in body fluids are attracting attention as diagnostic biomarkers that directly reflect neuropathological features within the brain. In particular, exosomal miRNAs (exomiRs) signatures are involved in AD pathogenesis, showing a different expression between patients and the healthy controls (HCs). However, low yield and high homologous nature impede the accuracy and reproducibility of exosome blood-based AD diagnostics. Here, we developed a programmable curved plasmonic nanoarchitecture-based biosensor to analyze exomiRs in clinical serum samples for accurate AD diagnosis. To allow the detection of exomiRs in serum at attomolar levels, nanospaces (e.g., nanocrevice and nanocavity) were introduced into the nanostructures to dramatically increase the spectral sensitivity by adjusting the bending angle of the plasmonic nanostructure through sodium chloride concentration control. The developed biosensor classifies individuals into AD, mild cognitive impairment (MCI) patients, and HCs through profiling and quantifying exomiRs. Furthermore, integrating analysis expression patterns of multiple exosomal biomarkers improved serum-based diagnostic performance (average accuracy of 98.22%). Therefore, precise, highly sensitive serum-derived exosomal biomarker detection-based plasmonic biosensor has a robust capacity to predict the molecular pathologic of neurodegenerative disease, progression of cognitive decline, MCI/AD conversion, as well as early diagnosis and treatment.

Enhancement of microalgal biomass productivity through mixotrophic culture process utilizing waste soy sauce and industrial flue gas.

Wastewater treatment plants are indispensable facilities, which emit a massive amount of greenhouse gases. To boost CO2 mitigation and wastewater treatment performance, mixotrophic microalgae cultivation using wastewater has recently been proposed. In this study, food industry wastewater (waste soy sauce) was applied to Chlorella sorokiniana UTEX 2714 cultivation. By using a medium with 20% (v/v) of 10-fold diluted soy sauce, the biomass and fatty acid methyl ester (FAME) productivity enhanced by 1.93 and 1.76 times, respectively. Biomass productivity increased up to 5.2 times when using medium with high soy sauce content under high-intensity light that inhibits cell growth in photoautotrophic environments. Furthermore, industrial flue gas treatment with wastewater was demonstrated by outdoor semi-continuous cultivation with 42% improved biomass production. Consequently, these results suggest that mixotrophic microalgal cultivation has great potential to address both climate change and water pollution while producing valuable products and can contribute to building a sustainable society.

Enhanced astaxanthin production of Haematococcus pluvialis strains induced salt and high light resistance with gamma irradiation.

This study was conducted to increase the productivity of biomass that contains high astaxanthin content by developing a mutant Haematococcus pluvialis strain with strong environmental tolerance. H. pluvialis has a low cell-growth rate and is vulnerable to stressors such as salinity or light intensity, which may hinder large-scale commercial cultivation. A mutant M5 strain selected through 5000-Gy gamma irradiation showed improved biomass and astaxanthin production under high-salinity and high-light intensity conditions. With enhanced SOD activity and overexpressed astaxanthin biosynthesis genes (lyc, crtR-b, bkt2), M5 demonstrated an increase in biomass and astaxanthin productivity by 86.70 % and 66.15 %, respectively compared to those of untreated cells. Also, the omega-3 content of M5 increased by 149.44 % under 40 mM CaCl2 compared to the untreated cells. Finally, even when subjected to high-intensity light irradiation for the whole life cycle, the biomass and astaxanthin concentration increased by 84.99 % and 241 %, respectively, compared to the wild-type cells.

The effects of acetate and glucose on carbon fixation and carbon utilization in mixotrophy of Haematococcus pluvialis.

The culture method using sodium acetate and glucose, widely used as organic carbon sources in the mixotrophy of Haematococcus pluvialis, was compared with its autotrophy. In the 12-day culture, mixotrophy using sodium acetate and glucose increased by 40.4% and 77.1%, respectively, compared to autotrophy, but the mechanisms for the increasing biomass were different. The analysis of the mechanism was divided into autotrophic and heterotrophic metabolism. The mixotrophy with glucose increased the biomass by directly supplying the substrate and ATP to the TCA cycle while inhibiting photosynthesis. Gene expressions related to glycolysis and carbon fixation pathway were confirmed in autotrophy and mixotrophy with glucose and acetate. The metabolism predicted in the mixotrophy with acetate and glucose was proposed via autotrophic and heterotrophic metabolism analysis. The mechanism of Haematococcus pluvialis under mixotrophic conditions with high CO2 concentration was confirmed through this study.

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.

Novel effective bioprocess for optimal CO2 fixation via microalgae-based biomineralization under semi-continuous culture.

In this study, the effects of microalgae-based biomineralization in a semi-continuous process (M-BSP) on biomass productivity and CO2 fixation rate were investigated. M-BSP significantly improved biomass production and CO2 fixation rate at the second stage of induction by sustaining relatively high photosynthetic rate without exposure to toxic substances (e.g., chlorellin) from aging cells using the microalgae Chlorella HS2. In conventional systems, cells do not receive irradiated light evenly, and many cells age and burst because of the long culture period. In contrast, in the M-BSP, the photosynthesis efficiency increases and biomass production is not inhibited because most of the cells can be harvested during shorter culture period. The accumulated biomass production and CO2 fixation rate of the HS2 cells cultured under M-BSP increased by 4.67- (25 ± 1.09 g/L) and 10.9-fold (30.29 ± 1.79 g/L day-1), respectively, compared to those cultured without the CaCl2 treatment.

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.

Sustainable microalgal biomass production in food industry wastewater for low-cost biorefinery products: a review.

Microalgae are recognized as cell factories enriched with biochemicals suitable as feedstock for bio-energy, food, feed, pharmaceuticals, and nutraceuticals applications. The industrial application of microalgae is challenging due to hurdles associated with mass cultivation and biomass recovery. The scale-up production of microalgal biomass in freshwater is not a sustainable solution due to the projected increase of freshwater demands in the coming years. Microalgae cultivation in wastewater is encouraged in recent years for sustainable bioeconomy from biorefinery processes. Wastewater from the food industry is a less-toxic growth medium for microalgal biomass production. Traditional wastewater treatment and management processes are expensive; hence it is highly relevant to use low-cost wastewater treatment processes with revenue generation through different products. Microalgae are accepted as potential biocatalysts for the bioremediation of wastewater. Microalgae based purification of wastewater technology could be a universal alternative solution for the recovery of resources from wastewater for low-cost biomass feedstock for industry. This review highlights the importance of microalgal biomass production in food processing wastewater, their characteristics, and different microalgal cultivation methods, followed by nutrient absorption mechanisms. Towards the end of the review, different microalgae biomass harvesting processes with biorefinery products, and void gaps that tend to hinder the biomass production with future perspectives will be intended. Thus, the review could claim to be valuable for sustainable microalgae biomass production for eco-friendly bioproduct conversions.

Waste mitigation and resource recovery from food industry wastewater employing microalgae-bacterial consortium.

Wastewater generated by the food industry is rich in nitrogen and phosphorus with possible presence of heavy metals. Physical and chemical methods of treatment, although effective, are expensive and may cause secondary environmental pollution damaging aquatic and human life. Traditional biological methods are eco-friendly and cost-effective but involve standalone microorganisms that pose risk of contamination and are not as effective. This review discusses the application of novel microalgal-bacterial consortium as a solution for the resource recovery and treatment of dairy, starch and aquaculture wastewater. Use of biofilm reactors containing anaerobic and aerobic sludge has shown 80-90% and > 90% COD and nutrient removal efficiency in treatment of dairy and starch processing wastewater, respectively. The treatment of aquaculture processing wastewater can be challenging due to high sality and requires salt-tolerant bacteria-microalgae consortium. In this regard, the identification of dominant microalgae and bacteria using 16S rRNA and 18S rRNA genes is recommended.

Three-dimensional hierarchical plasmonic nano-architecture based label-free surface-enhanced Raman spectroscopy detection of urinary exosomal miRNA for clinical diagnosis of prostate cancer.

The urinary exosomal miRNAs are recently emerging prostate cancer (PC)-associated biomarkers for the early-stage diagnosis and prognosis due to their non-invasiveness, inherent stability and the representation of the status of the originated cells. However, developing a urinary exosomal miRNA detection method with high accuracy is challenging because of the low abundance and high sequence homology of miRNAs. Herein, we present a quantitative and label-free miRNA sensing platform using surface-enhanced Raman scattering (SERS) based on three-dimensional (3D) hierarchical plasmonic nano-architecture to detect urinary exosomal miRNAs. This hierarchical nanostructure is constructed by self-assembly between target-complementary DNA probes-conjugated gold nanoparticles and head-flocked gold nanopillars in the presence of the target miRNAs, creating numerous 3D plasmonic hot-spots inducing exceedingly high amplification of SERS signals. This 3D SERS biosensor achieved ∼10 aM detection limits for the target miRNAs (miR-10a and miR-21), which is over 1000-fold more sensitive than previously reported miRNA sensors without the requirement of any labelling or pre-treatment steps. Finally, the clinical validation using urinary samples revealed that our 3D SERS sensor discriminates PC patients from healthy control with high diagnostic accuracy (0.93) based on the differential expression level of urinary exosomal miRNAs. These outputs demonstrate that our SERS sensor based on 3D hierarchical nano-architecture can offer facile, accurate and rapid methods to measure miRNA expression and is helpful for the diagnosis of various diseases.

Macular pigment-enriched oil production from genome-edited microalgae.

BACKGROUND: The photosynthetic microorganism Chlamydomonas reinhardtii has been approved as generally recognized as safe (GRAS) recently, this can excessively produce carotenoid pigments and fatty acids. Zeaxanthin epoxidase (ZEP), which converts zeaxanthin to violaxanthin, and ADP-glucose pyrophosphorylase (AGP). These are key regulating genes for the xanthophyll and starch pathways in C. reinhardtii respectively. In this study, to produce macular pigment-enriched microalgal oil, we attempted to edit the AGP gene as an additional knock-out target in the zep mutant as a parental strain. RESULTS: Using a sequential CRISPR-Cas9 RNP-mediated knock-out method, we generated double knock-out mutants (dZAs), in which both the ZEP and AGP genes were deleted. In dZA1, lutein (2.93 ± 0.22 mg g-1 DCW: dried cell weight), zeaxanthin (3.12 ± 0.30 mg g-1 DCW), and lipids (450.09 ± 25.48 mg g-1 DCW) were highly accumulated in N-deprivation condition. Optimization of the culture medium and process made it possible to produce pigments and oil via one-step cultivation. This optimization process enabled dZAs to achieve 81% higher oil productivity along with similar macular pigment productivity, than the conventional two-step process. The hexane/isopropanol extraction method was developed for the use of macular pigment-enriched microalgal oil for food. As a result, 196 ± 20.1 mg g-1 DCW of edible microalgal oil containing 8.42 ± 0.92 mg g-1 lutein of oil and 7.69 ± 1.03 mg g-1 zeaxanthin of oil was produced. CONCLUSION: Our research showed that lipids and pigments are simultaneously induced in the dZA strain. Since dZAs are generated by introducing pre-assembled sgRNA and Cas9-protein into cells, antibiotic resistance genes or selective markers are not inserted into the genome of dZA, which is advantageous for applying dZA mutant to food. Therefore, the enriched macular pigment oil extracted from improved strains (dZAs) can be further applied to various food products and nutraceuticals.

Design and applications of photobioreactors- a review.

There has been increasing attention in recent years on the use of photobioreactors for various biotechnological applications, especially for the cultivation of microalgae. Photobioreactors-based production of photosynthetic microorganisms furnish several advantages as minimising toxicity and providing improved conditions. However, the designing and scaling-up of photobioreactors (PBRs) remain a challenge. Due to huge capital investment and operating cost, there is a deficiency of suitable PBRs for development of photosynthetic microorganisms on large-scale. It is, therefore, highly desirable to understand the current state-of-the-art PBRs, their advantages and limitations so as to classify different PBRs as per their most suited applications. This review provides a holistic overview of the discreet features of diverse PBR designs and their purpose in microalgae growth and biohydrogen production and also summarizes the recent development in use of hybrid PBRs to increase their working efficiency and overall economics of their operation for the production of value-added products.