Microfluidic chemostatic bioreactor for high-throughput screening and sustainable co-harvesting of biomass and biodiesel in microalgae.

As a renewable and sustainable source for energy, environment, and biomedical applications, microalgae and microalgal biodiesel have attracted great attention. However, their applications are confined due to the cost-efficiency of microalgal mass production. One-step strategy and continuous culturing systems could be solutions. However, current studies for optimization throughout microalgae-based biofuel production pipelines are generally derived from the batch culture process. Better tools are needed to study algal growth kinetics in continuous systems. A microfluidic chemostatic bioreactor was presented here, providing low-bioadhesive cultivations for algae in a cooperative environment of gas, nutrition, and temperature (GNT) involved with high throughput. The chip was used to mimic the continuous culture environment of bioreactors. It allowed simultaneously studying of 8 × 8 different chemostatic conditions on algal growth and oil production in parallel on a 7 × 7 cm2 footprint. On-chip experiments of batch and continuous cultures of Chlorella. sp. were performed to study growth and lipid accumulation under different nitrogen concentrations. The results demonstrated that microalgal cultures can be regulated to grow and accumulate lipids concurrently, thus enhancing lipid productivity in one step. The developed on-chip culturing condition screening, which was more suitable for continuous bioreactor, was achieved at a half shorter time, 64-times higher throughput, and less reagent consumption. It could be used to establish chemostat cultures in continuous bioreactors which can dramatically accelerate the development of renewable and sustainable algal for CO2 fixation and biosynthesis and related systems for advanced sustainable energy, food, pharmacy, and agriculture with enormous social and ecological benefits.

A microfluidic droplet array demonstrating high-throughput screening in individual lipid-producing microalgae.

Microalgae are a group of photoautotrophic microorganisms which could use carbon dioxide for autosynthesis. They have been envisioned as one of the most prospective feedstock for renewable oil. However, great endeavors will still be needed to increase their economic feasibility. The screening of competitive species and suitable culture conditions are such issues. To greatly accelerate these rather laborious steps and also improve their experimental lump-sum-manner, we developed a microfluidic droplet-based 2 × 103 resolution "identification card", which allowed high throughput real-time monitoring of individual algae among population. A novel fluid-blocking-based droplet generating and trapping performance were integrated in the platform which made it excellent in operational simplicity, rapidity and stability and full of the potentials in single-cell-isolation/screening. The developed platform was successfully used to screen three unicellular algae, namely, Isochrysis zhanjiangensis, Platymonas subcordiformis and Platymonas helgolandica var. tsingtaoensis. In situ bioassays of the lipid accumulation and cell proliferation at single cell level for interspecies comparison were possible. Furthermore, lipid-producing inhomogeneity was demonstrated among cells in the same specie and batch. Nitrogen stress condition can be identified that induce positive-skewed frequency distribution of lipid content, even among individual inhomogeneous cells over the typically used culture condition.

Instrumentation-Compact Digital Microfluidic Reaction Interface-Extended Loop-Mediated Isothermal Amplification for Sample-to-Answer Testing of Vibrio parahaemolyticus.

Vibrio parahaemolyticus is usually spread via consumption of contaminated seafood and causes vibriosis. By combination of digital microfluidic (DMF) and loop-mediated isothermal amplification (LAMP), we provided an automated instrumentation-compact DMF-LAMP device for sample-to-answer detection of V. parahaemolyticus. For the first time, how much the proper mixing might facilitate the DMF-LAMP process is explored. The results illustrated that increasing the number of flow configurations and decreasing the fluid-reversibility will extend the interfacial surface available for diffusion-based mass transfer within a droplet microreactor, thus contributing to the overall amplification reaction rate. Noticeably, the DMF-LAMP amplification plateau time is shortened by proper mixing, from 60 min in static mixing and traditional bulk LAMP to 30 min in 2-electrode mixing and 15 min in 3-electrode mixing. The device achieved much higher detection sensitivity (two copies per reaction) than previously reported devices. V. parahaemolyticus from spiked shrimps is detected by Q-tip sampling associated with 3-electrode mixing DMF-LAMPs. The detectable signal occurs within only 3 min at a higher concentration and, at most, is delayed to 18 min, with a detection limit of <0.23 × 103 CFU/g. Thus, the developed DMF-LAMP device demonstrates potential for being used as a sample-to-answer system with a quick analysis time, high sensitivity, and sample-to-answer format.

A three-dimensional pinwheel-shaped paper-based microfluidic analytical device for fluorescence detection of multiple heavy metals in coastal waters by rational device design.

Here, we present the rational design of a pinwheel-shaped three-dimensional microfluidic paper-based analytical device (3D-µPAD) for specific, sensitive and multiplexed detection of heavy metals in coastal waters. A more homogeneous permeation of fluids along the chip than common design, even under unskilled performance, has been achieved by the elaborate chip design of the hydrostatic balancing inlet port and uniformly stressed reversible sealing. With the combination of ion imprinted polymer grafted CdTe quantum-dots and fluid accumulation pad, 4 metals (Cu2+, Cd2+, Pb2+, and Hg2+) in 1 analysis and 25-fold enrichment for each metal can be simultaneously performed within 20 min, with detection limits of 0.007-0.015 µg/L. It has the ability to selectively recognize these 4 metals in mixtures and immunizing to interferences from components found in coastal waters, which provided results that were in agreement with values gained from atomic absorption. The inexpensive and portable nature as well as the highly sensitive and flexible performance of the new developed 3D-µPAD could make it attractive as an on-site testing approach for marine environmental monitoring.

An integrated digital microfluidic bioreactor for fully automatic screening of microalgal growth and stress-induced lipid accumulation.

Algae are the promising feedstock of biofuel. The screening of competent species and proper fertilizer supply is of the most important tasks. To accelerate this rather slow and laborious step, we developed an integrated high-throughput digital microfluidic (DMF) system that uses a discrete droplet to serve as a microbioreactor, encapsulating microalgal cells. On the basis of fundamental understanding of various droplet hydrodynamics induced by the existence of different sorts of ions and biological species, incorporation of capacitance-based position estimator, electrode-saving-based compensation, and deterministic splitting approach, was performed to optimize the DMF bioreactor. Thus, it enables all processes (e.g., nutrient gradient generation, algae culturing, and analyzing of growth and lipid accumulation) occurring automatically on-chip especially in a high-fidelity way. The ability of the system to compare different microalgal strains on-chip was investigated. Also, the Chlorella sp. were stressed by various conditions and then growth and oil accumulation were analyzed and compared, which demonstrated its potential as a powerful tool to investigate microalgal lipid accumulation at significantly lower laborites and reduced time.

RTA and LANA Competitively Regulate let-7a/RBPJ Signal to Control KSHV Replication.

The recombination signal binding protein for immunoglobulin kappa J region (RBPJ) has a dual effect on Kaposi's sarcoma-associated herpesvirus (KSHV) replication. RBPJ interaction with replication and transcription activator (RTA) is essential for lytic replication, while the interaction with latency-associated nuclear antigen (LANA) facilitates latent infection. Furthermore, our previous study found that LANA decreased RBPJ through upregulating miRNA let-7a. However, it is unclear whether RTA regulates the expression of RBPJ. Here, we show RTA increases RBPJ by decreasing let-7a. During KSHV replication, the RBPJ expression level was positively correlated with the RTA expression level and negatively correlated with the LANA expression level. The let-7a expression level was inverse to RBPJ. Knockdown of RBPJ inhibited the self-activation of RTA promoter and LANA promoter and weakened LANA's inhibition of RTA promoter. Collectively, these findings indicate that RTA and LANA compete for let-7a/RBPJ signal to control the KSHV replication. Regulating the RBPJ expression level by RTA and LANA plays an important role during KSHV replication.

Microenvironment-Controlled Micropatterned Microfluidic Model (MMMM) for Biomimetic In Situ Studies.

Attachment of trophozoites to the intestine is an indispensable step for Giardia's survival and pathogenicity in almost 280 million infections worldwide each year. However, the analysis of the attachment mechanism is difficult due to the lack of methods that can create a favorable microaerobic atmosphere. Herein, we developed an osmotic-pressure, pH, excretion, nutrition, gas, ionic-strength, flow-rate, and temperature microenvironment-controlled micropatterned microfluidic model to simulate the in vivo microenvironment to study in situ the stress applied to Giardia in the intestinal tract. We designed three nonbiological surfaces with stagger arrangement manners and integrated them with a resistance microfluidic network to split Giardia-attaching forces ingeniously and developed the term "attaching contribution rate" (ACR) to describe their corresponding contributions. Our study shows that the total attaching force measured is 49.58 Pa, with three components being 22.66 Pa (suction force), 12.52 Pa (clutching force), and 14.4 Pa (combined electrostatic and van der Waals force), respectively, with ACRs being 46%, 25%, and 29%, respectively. By decomposing the attaching force and analyzing each force component and their structure and composition basis, whole profiles of the attachment mechanisms were revealed. Our method enables the analysis of the surface attachment mechanisms and their ACRs for Giardia.

A digital microfluidic diluter-based microalgal motion biosensor for marine pollution monitoring.

Marine pollution and monitoring have received more and more concern in recent years. Herein, a fully automatic whole-algae biosensor was designed for low-cost and fast detection of toxic contaminants in seawater. It consists of a digital microfluidic (DMF) diluter chip, an actuation element, a detector element, and a microalgae bioreporter. A feedback-control protocol based on charging-time compensation was introduced. It ensures precise actuation of the droplet with diverse salty concentrations and contents in the marine environment. The two-mixer cross-split dilution engine increases the accuracy of droplet dispensing and concentration diluting. By selecting motility of P. subcordiformis as the sensor signal, the developed biosensor showed good sensitivity and robustness for a wide range of salinity (10-37‰), temperature (0-25 °C), light levels (0-325 µmol photons m-2 s-1), and cell density factor (1.0-4.0). The biosensor responses were examined in the presence of copper, lead, phenol, and nonylphenol (NP). In all cases, toxic responses (i.e. dose-related inhibition of algal motion) were detected with the detection limits of 0.65  µmol.L-1, 1.90  µmol.L-1, 2.85 mmol.L-1, and 5.22  µmol.L-1 respectively. These results were obtained in a much shorter time (2 h for our biosensor vs. 24 h-10 d for growth inhibition test) and the data are consistent with previous classical studies. We thus developed a simple, rapid, and adaptable system for marine routine monitoring and early-warning detection for lab and on-site applications.

An efficient protocol to use feedback-controlling digital microfluidic fluorimetric sensor for detection of mercury (II) in coastal seawaters.

Mercury ion is a highly toxic anthropogenic pollutants and has serious well-known effects on human. There is an ever-growing demand for convenient detection of mercury driven contaminants in environment, including coastal seawater. However, most of the reported methods are instrument-based and are not easy for portable detection. Our protocol described an efficient Digital Microfluidics method for detecting mercury in coastal seawater samples. It combined the miniaturization/automation potential of digital microfluidics and the sensitivity of fluorescence probe. To overcome a potential risk of driven failure, induced by diversity ion ingredients in seawater, a feedback control loop was included into control system. The method showed satisfied stability and selectivity in Hg sensing under high salinity condition, with the sensitivity of Hg2+ at the parts-per-billion level and total testing time of less than 20 s. With the advantages of being fast, amenable to automation and low cost, this protocol is promising for the formation of simple and rapid sensor device, especially for a routine monitoring and emergency detection of Hg/or other metals in coastal waters.

A feedback-controlling digital microfluidic fluorimetric sensor device for simple and rapid detection of mercury (II) in costal seawater.

By combination of miniaturization potential of digital microfluidics (DMF) and sensitivity of fluorescence probe, an integrated sensor device has been initially constructed for mercury detection in coastal waters. The actuation feature of the detecting target, seawater droplet, which remains unclear, was basically explored. To overcome a potential risk of driven failure, induced by diversity ion ingredients in seawater, a feedback control loop was included into control system. Analyzing method for coastal waters was well established on DMF, which showed satisfied stability and selectivity in Hg sensing under high salinity condition, with the sensitivity of Hg2+ at the parts per billion level and total testing time less than 20s. With the advantages of being fast, amenable to automation and low cost, this device is promising for the formation of simple and rapid sensor device, especially for a routine monitoring and emergency detection of Hg/or other metals in coastal waters.

Latency-associated nuclear antigen inhibits lytic replication of Kaposi's sarcoma-associated herpesvirus by regulating let-7a/RBPJ signaling.

Latency-associated nuclear antigen (LANA) is the key factor in the establishment and maintenance of latency of Kaposi's sarcoma-associated herpesvirus (KSHV). A cellular protein, recombination signal binding protein for immunoglobulin kappa J region (RBPJ), is essential for the lytic reactivation of KSHV. However, whether RBPJ expression is regulated by KSHV is not clear. Here, we show that LANA upregulates let-7a and its primary transcripts in parallel with its reduction of RBPJ expression. An increase in notch intracellular domain (NICD) and the downregulation of NF-κB and LIN28B contribute to the upregulation of let-7a by LANA. Let-7a represses RBPJ expression by directly binding the 3' untranslated region of RBPJ. Let-7a overexpression or RBPJ knockdown led to a dose- and time-dependent inhibition of lytic reactivation of KSHV. Collectively, these findings support a model wherein LANA inhibits the lytic replication of KSHV by regulating let-7a/RBPJ signaling.

Computer-aided design of microfluidic resistive network using circuit partition and CFD-based optimization and application in microalgae assessment for marine ecological toxicity.

We present an automatic design process for microfluidic dilution network towards marine ecological toxicity assessment on microalgae. Based on the hydraulic-electric circuit analogy, we defined an abstract specification using computer-aided designing system. Several approaches, especially circuit partition, were applied to minimize design effort. Computational fluid dynamics (CFD) simulation was exploited to convert the electrics specification to fabrication model. We automatically designed the combinational-mixing-serial dilution microfluidics to generate parallel stepwise gradients for mixing chemicals (binary/ternary/quaternary mixture) using the present algorithm. We critically discussed design rules and evaluated the microfluidic performance by colorimetric analysis. To examine whether these microfluidic chips can be used for toxicity test on microalgae, single and joint toxic effects of heavy metals (copper, mercury, zinc, and cadmium) were examined on line. In all cases, dose-related toxic responses were successfully detected. These results provided a solution for designing resistive network using circuit partition and CFD-based optimization and a route to develop a promising user-friendly alternative for microalgae bioassays as well as cell-based screening experiments in risk assessment.

Development of Microfluidic Dilution Network-Based System for Lab-on-a-Chip Microalgal Bioassays.

Because of the crucial ecological significance of microalgae, microalgal bioassays have become one of the most demanding tests from all classic aquatic toxicity tests in regulatory frameworks. However, conventional algal tests tend to be lab-intensive and time- and space-consuming, and they have not been utilized to their full potential for routine toxicity assessments. Microfluidics should be a user-friendly alternative. Particularly, dilution to generate gradients that are appropriate for screening experiments can be precisely attained by microfluidic network in a simple and cost-/time-/space-saving way. Here, we demonstrate a microfluidics series toward routine microalgal bioassays, including pretest, single, and joint toxicity test. The chip mainly consists of upstream dilution network (single serial dilution module (logarithmic/linear gradient generator) or multiple (binary/ternary/quaternary) mixing serial dilution module) and downstream diffusible culturing module. It allows the processes of chemical liquid dilution and diffusion, microscale microalgal culture, cell stimulation, and online screening to be integrated into a single device. Electric theorems with the aid of EDA (electronic design automation) simulation were innovatively introduced to minimize design effort for such systems. Using the device, microalgae were successfully cultured and stressed on-chip. The simple assay provides multibiological trait assessments of cell division rate, autofluorescence, esterase activity, and mobile capacity. This work showed promise in developing a high-throughput microfluidic platform for microalgal bioassays as well as lab-on-a-chip screening experiments in the cell-based quantitative assessment of environmental health risks.

Long-term effects of combined divalent copper and tetracycline on the performance, microbial activity and community in a sequencing batch reactor.

The long-term effects of combined divalent copper (Cu(II)) and tetracycline (TC) on the performance, microbial activity and community in a sequencing batch reactor (SBR) were investigated. The addition of Cu(II), TC or mixed Cu(II)/TC caused the decrease of the organics and nitrogen removal efficiencies, and their decreased degrees were the lowest at the addition of mixed Cu(II)/TC. The increase of mixed Cu(II)/TC concentrations in the influent did not change the antagonistic effects between Cu(II) and TC on nitrifying and denitrifying activities. Nitrifiers had higher tolerances to Cu(II), TC and mixed Cu(II)/TC than denitrifiers. Compared to the addition of Cu(II) or TC alone, the microbial community richness was higher at the addition of mixed Cu(II)/TC, while the microbial community diversity was lower. The increased protein (PN) in extracellular polymeric substances (EPS) was a protective response of bacteria to Cu(II), TC and mixed Cu(II)/TC.

Measurement of Giardia lamblia adhesion force using an integrated microfluidic assay.

The mechanisms how Giardias attach to the intestinal epithelium remain unclear. None of the methods currently being used to measure the attachment force could provide a continuous nutrition supply and a micro-aerobic atmosphere to the Giardia. Besides, they are all labor-intensive. In the present research, a microfluidic method based on electric circuit analogy was developed. The input fluid flowed through the inlet channel with different lengths and was distributed in four assay chambers. Shear force gradients were generated in chambers, too. This allowed an easy control of fluids and the shear forces. Most importantly, the shear stress large enough to detach Giardia could be generated in laminar flow regime. Moreover, analysis could be accomplished in one single test. By applying inlet flow rates of 30, 60, and 120 µL ml-1, shear force gradients ranging from 19.47 to 60.50 Pa were generated. The adhesion forces of trophozoites were analyzed and the EC50 of the force that caused 50% trophozoites detachment was calculated as 36.60 Pa. This paper presents a novel method for measurement of Giardia adhesion force. Graphical Abstract Measurement of Giardia adhesion force. Various of flow rates were applied to generate different shear forces and Giardia trophozoites remaining attached were counted (a-c). The percentages of attachment vs shear stress were plotted and the EC50 of adhesion force was calculated (d).

Giardia duodenalis GlSir2.2, homolog of SIRT1, is a nuclear-located and NAD(+)-dependent deacethylase.

Sir2 family proteins are highly conserved and catalyze Nicotinamide Adenine Dinucleotide (NAD(+))-dependent protein deacetylation reaction that regulates multiple cellular processes. Little is known about Sir2 family proteins in Giardia. In this research, Sir2 homologs of Giardia were Phylogenetically analyzed. GL50803_10707 (GlSIR2.2) showed strong homology to SIRT1 and was the only parasite SIRT1 homolog being reported to date. Recombinant GlSIR2.2 (rGlSIR2.2) was expressed and purified. The renaturied recombinant protein showed a typical NAD-dependent protein deacetylase activity that could be inhibited by nicotinamide, with IC50 of 4.47 mM rGlSIR2.2 displayed deacetylase activity under varied NAD(+), with Km, kcat and kcat/Km values of 31.71 µM, 1.4 × 10(-3) s(-1), and 4.42 × 10(-5) µM(-1) s(-1). Similarly, the steady-state kinetic parameters with varied ZMAL, yielded Km, kcat and kcat/Km values of 96.89 µM, 4.7 × 10(-3) s(-1), and 4.85 × 10(-5) µM(-1) s(-1). Anti-rGlSIR2.2 serum was used to probe subcellular localization of GlSIR2.2 and strong staining was found predominantly in the nucleus. So we demonstrated that GlSIR2.2 was a SIRT1-like, nuclear-located, NAD(+)-dependent deacetylase. This is the first report of deacetylase activity of Sir2 family protein in Giardia.

An on-chip pollutant toxicity determination based on marine microalgal swimming inhibition.

We report the use of microalgal swimming behavior as a sensor signal integrated into microfluidics for a rapid and high-throughput determination of pollutant toxicity. There are two types of chip. A poly(dimethylsiloxane) (PDMS) 12-well chip, used for optimization of experimental conditions (i.e. light level, temperature, initial cellular density and exposure time), can perform twelve parallel tests simultaneously. In a concentration gradient generator (CGG) chip, a CGG connected with diffusible chambers enables a large number of dose-response bioassays to be performed in a simple way. Microalgal swimming was set as a microfluidic bioassay signal and was evaluated as swimming manner, motile percentage (%MOT), curvilinear velocity (VCL), average path velocity (VAP) and straight line velocity (VSL). Under optimized physical conditions, the toxicities of Cu, Pb, phenol and nonylphenol (NP) towards four mobile marine microalgae, Platymonas subcordiformis, Platymonas helgolandica var. tsingtaoensis, Isochrysis galbana and Isochrysis zhanjiangensis sp. nov, were investigated. In all cases, a toxic response (i.e. a dose-related inhibition of swimming) was detected, and a time of only 2 h was needed to predict EC50 values. The 2h-EC50s showed that I. galbana was the most tolerant and that P. subcordiformis was one of the most sensitive. Based on the relative motile percentage data, the EC50 values for Cu of I. galbana and P. subcordiformis were 6.04 and 1.67 µM, respectively, while for Pb the EC50 values were 15.30 and 3.87 µM, for phenol the EC50 values were 8.69 and 6.08 mM, and for NP the EC50 values were 29.65 and 14.47 µM, respectively. Taking into account all the swimming inhibition parameters, MOT provided more sensitive EC results. The sensitivity differences between the velocity parameters (VCL, VAP and VSL) were ascribed to differences in swimming manner of the different classes of microalgae.

Microfluidic aqueous two-phase extraction of bisphenol A using ionic liquid for high-performance liquid chromatography analysis.

An aqueous two-phase microfluidics (ATPM) method suitable for selective extraction of bisphenol A (BPA) in aqueous samples was developed, and a functional ionic liquid of N, N, N-trioctyl ammonium propionate (TOAP) was specially employed for the formation of a parallel flow system. Based on the analytical model, we optimized the chip design into branch-connection length pattern to achieve maximum extraction efficiency (φ max) and ensure phase separation. In combining the design flexibility and ideal reaction activity of extractant (TOAP), the developed ATPM enabled a selective and effective extraction of BPA (φ max of 95% within 2 s) from phenol derivatives. Meanwhile, the total operation time and ionic liquid consumption of the microfluidic extraction were only 2.5 min and 5 µl, respectively. The ATPM can be run at normal pH and room temperature and showed no interferences from components found in tap or beach water. To be noted, this specific extraction system was applied in real water samples; the recoveries of standard addition for all water samples spiked with BPA were from 96 to 110%. Finally, successful reuse of the chip was also realized. In all cases, the developed microfluidic chip was proven to be useful as an effective and low consumption approach in extracting BPA and should be expanded as a "green" preparative method for high-performance liquid chromatography (HPLC) analysis.

[Application of microfluidics in aquatic environmental pollution analysis].

Recently, a new type of chip technology, microfluidics, has received global attention for its rapid analysis speed, low reagent consumption, small size and simple operation, etc. Based on a micro-channel network and supported by a Micro-Electro-Mechanic System (MEMS), this technology integrates all the functions of a laboratory into one small piece of chip, which is called "lab on the chip". This paper presented a brief introduction about microfluidics and its representative developments. Future prospects in the aspects of instrument miniaturization, system integration, chip materials, and detection techniques, as well as the implementation of microfluidics in aquatic environmental pollutant analysis were thoroughly discussed. Some problems faced now were put forward. With the rapid progress in the microfluidics, a universal low-cost microchip capable of high speed multi-channel detection and integrated with many kinds of detection methods would be the research focus in the future.

Marine phytoplankton motility sensor integrated into a microfluidic chip for high-throughput pollutant toxicity assessment.

A microfluidic chip was designed to assess the toxicity of pollutants in a high-throughput way by using marine phytoplankton motility as a sensor signal. In this chip, multiple gradient generators (CGGs) with diffusible chambers enable large scale of dose-response bioassays to be performed in a simple way. Two mobile marine phytoplankton cells were confined on-chip and stimulated by 8 concentrations (generated by CGG) of Hg, Pb, Cu and phenol singly, as well as Cu and phenol jointly. CASA system was used to characterize motility by motile percentage (%MOT), curvilinear velocity (VCL), average path velocity (VAP) and straight line velocity (VSL). In all cases, dose-dependent inhibitions of motility were observed. In the present system, only 2h was needed to predict EC50. Thus, the developed microfluidic chip device was proved to be useful as a rapid/simple and high-throughput test method in marine pollution toxicity assessment.