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

The gut microbial community is critical for the host immune system, and in recent years, it has been extensively studied in vertebrates using ‘omic’ technologies. In contrast, knowledge about how the interactions between water temperature and diet affect the gut microbiota of marine invertebrates that do not thermoregulate is much less studied. In the present study, the effect of elevated seawater temperature and diet (Isochrysis zhanjiangensis and Platymonas helgolandica var. tsingtaoensis) on the gut microbial community of the commercial mussel, Mytilus coruscus, was investigated. The 16S rRNA gene sequencing was used to characterize the microbial community in M. coruscus gut. The mortality of M. coruscus exposed to a high water temperature (31°C) increased after 3 days and the diversity of the bacterial community in the gut of live M. coruscus was significantly reduced. For example, the abundance of Bacteroides (Bacteroidetes) and norank_Marinilabiaceae (Bacteroidetes) increased in the gut of M. coruscus fed I. zhanjiangensis. In M. coruscus fed P. helgolandica, the abundance of Arcobacter (Proteobacteria) and norank_Marinilabiaceae increased and the abundance of unclassified_Flavobacteriaceae (Bacteroidetes) decreased. The results obtained in the present study suggest that high temperatures favored the proliferation of opportunistic bacteria, including Bacteroides and Arcobacter, which may increase host susceptibility to disease. Microbial community composition of the gut in live M. coruscus was not impacted by the microalgal diet but it was modified in the group of mussels that died. The present study provides insight into the potential effects on the gut microbiome and mussel–bacteria interactions of rising seawater temperatures.

A polydimethylsiloxane microfluidic chip has been developed for the estimation of toxic heavy metals based on measurement of mobility of marine microalgae. The chip is mainly composed of an upstream concentration gradient generator and a downstream perfusion-based chemotatic module. The processes of toxic liquid dilution and diffusion, microalgal culturing, cell stimulation, and online screening can be integrated in this chip, which makes it an attractive approach to simplify toxicity testing procedures. The microalgal motility was adopted as a microfluidic bioassay signal and was evaluated as the percentage of motile cells, curvilinear velocity, average path velocity, and straight line velocity. Two mobile marine microalgae, Platymonas subcordiformis and Platymonas helgolandica var. tsingtaoensis, were confined in the chemotatic module and stimulated by the eight concentration gradients of Cu and Cd generated by the concentration gradient generator. In all cases, a toxic response was detected (i.e., a dose-related inhibition of motility was observed). Only 1.5 h was needed to predict EC(50) values. Thus, the microfluidic chip developed was proved to be useful as a simple and rapid approach in heavy metal detection and might be expanded as a conventional test method in environmental toxicity assessment.

The objective of this study was to examine the effect of benzo[a]pyrene (BaP) on the detoxification and antioxidant systems of two microalgae, Isochrysis zhanjiangensis and Platymonas subcordiformis. In our study, these two algae were exposed to BaP for 4 days at three different concentrations including 0.5 μg L−1 (low), 3 μg L−1 (mid) and 18 μg L−1 (high). The activity of detoxification enzymes, ethoxyresorufin O-deethylase (EROD) and glutathione S-transferase (GST) increased in P. subcordiformis in all BaP-treated groups. In I. zhanjiangensis, the activity of these two enzymes increased at the beginning of exposure, and then decreased in the groups treated with mid- and high BaP. The activity of antioxidant enzyme superoxide dismutase (SOD) increased in I. zhanjiangensis in all BaP-treated groups, and then decreased in high BaP-treated group, while no significant change was observed in P. subcordiformis. The activity of antioxidant enzyme catalase (CAT) increased in I. zhanjiangensis and P. subcordiformis in all BaPtreated groups. The content of malondialdehyde (MDA) in Isochrysis zhanjiangensis increased first, and then decreased in high BaP-treated group, while no change occurred in P. subcordiformis. These results demonstrated that BaP significantly influenced the activity of detoxifying and antioxidant enzymes in microalgae. The metabolic related enzymes (EROD, GST and CAT) may serve as sensitive biomarkers of measuring the contamination level of BaP in marine water.

Industrial farming is an alternative mode for Pinctada maxima juvenile cultivation to avoid mass mortality caused by natural disasters. Suitable and enough food is crucial for successful industrial bivalve farming. To investigate the feasibility of live microalga instead of spray-dried microalgal powder in P. maxima juvenile industrial farming, this study replaces a positive control live microalgal diet [Isochrysis zhanjiangensis (L-iso) and Platymonas subcordiformis (L-pla)] with spray-dried I. zhanjiangensis powder (P-iso) and P. subcordiformis powder (P-pla). Continuous feeding trials (30 days) were conducted on the P. maxima juvenile (1.2008 ± 0.0009 g initial weight and 30.12 ± 0.05 mm initial shell length), under laboratory conditions. Survival, growth performance, and intestinal microbial community were studied and compared across the groups. Results showed that survival rate (SR) did not differ significantly across the groups (ranged from 84 to 86%, P > 0.05). The growth performance in spray-dried microalgal groups, including total weight (TW), shell height (SH), absolute growth rate (AGR), and relative growth rate (RGR) for SH and TW, was slightly lower than that in live microalgal groups, while the activities of pepsin (PES), amylase (AMS), and lipase (LPS) were significantly higher (P < 0.05). The best growth performance was observed in the L-iso group, followed by the L-pla group. A 16S rRNA-based sequencing revealed that Proteobacteria was the dominant phylum in P. maxima juvenile intestinal bacterial community under controlled conditions, which accounted for 62–82% across groups. The intestinal bacteria at the genus level were more sensitive to diets, whereas Burkholderia was the dominant genus in both L-iso (66.52 ± 6.43%) and L-pla groups (54.00 ± 5.66%), while Mycoplasma, Alphaproteobacteria, and Oxyphotobacteria were in both P-iso and P-pla groups. The P-pla group got higher ACE, Chao1, and Simpson and Shannon indices (P < 0.05). The above results suggested that the spray-dried P-iso and P-pla can serve as substitutes for live microalga in P. maxima juvenile industrial farming under controlled conditions. The finding in this study provides basic data to optimize industrial farming technology and healthy management for P. maxima juvenile.

The razor clam (Sinonovacula constricta) is a bivalve species living in the lower to mid intertidal zones along the coasts of China, Japan and Korea. In this study, the effects of temperature, microalgae species and concentration on the absorption efficiency (AE) of cultured adult S Constricta fed with six species of microalgae, including Chlorella sp., Pavlova viridis, Nitzschia closterium f. minutissima, Platymonas subcordiformis, Nannochloropsis oculata and Isochrysis zhanjiangensis was examined. The clams were exposed to three different temperatures (15, 20 and 25°C) and five microalgae concentrations (0.16, 0.27, 0.37, 0.45 and 0.53 mg L−1). The results indicated that under different temperatures, there is significant difference (P < 0.05) in AE and the efficiency peaks at the water temperature of 20°C. Under different microalgae concentrations, the absorption efficiencies were also significantly different (P < 0.05) and there was a negative correlation between AE and microalgae concentration. At different combinations of temperature and microalgae concentration, the absorption efficiencies of Chlorella sp. and N. oculata were lower than those of other microalgae. The interaction of temperature and microalgae concentration affected the AE significantly (P < 0.05).

Pseudodiaptomus dubia is a calanoid copepod abundant in the mariculture ponds of southern China. However, our understanding of the population dynamics of P. dubia in aquaculture ponds is limited. In this study, groups of larval P. dubia were each fed a different microalgal species, and the effects of these different diets on development, survival, and reproduction were assessed. The five microalgae used were species common in aquaculture farms in China, and included two chlorophytes (Chlorella saccharophila and Platymonas subcordiformis), one golden microalga (Isochrysis zhanjiangensis), and two diatoms (Chaetoceros muelleri and Cyclotella meneghiniana). Our results indicated that C. saccharophila was not a suitable food for larval P. dubia, as all larvae fed this microalga died at stage III (as copepodites). The survival rates of P. dubia larvae fed C. muelleri, I. zhanjiangensis, and P. subcordiformis were significantly higher than that of larvae fed C. meneghiniana. In the adult stage, copepods fed C. muelleri, I. zhanjiangensis, and C. meneghiniana produced more nauplii (430–566 nauplii/female), had higher intrinsic growth rates (0.2–0.253/d), and better longevity (59–60 days) than those fed P. subcordiformis. Our results therefore suggest that P. dubia has different nutritional needs and food preferences at different life stages. For example, P. subcordiformis was suitable for developing larvae but not for breeding adults, while C. meneghiniana was suitable for breeding adults but not for developing larvae. Both C. muelleri and I. zhanjiangensis were excellent foods for P. dubia throughout the entire life cycle.

To understand the influences of environmental conditions on the performance of pearl oyster spat, we conducted four experiments to evaluate separately the effects of salinity (21, 24, 27, and 30), diet (Isochrysis zhanjiangensis, Platymonas subcordiformis, Chlorella; 50% I. zhanjiangensis/50% P. subcordiformis, 50% I. zhanjiangensis/50% Chlorella, and 50% P. subcordiformis/50% Chlorella), diet availability (high, medium and low), and rearing site (hatchery and sea) on the growth and survival of pearl oyster Pinctada maxima spat. Results showed that environmental conditions exerted significant effects on the growth of P. maxima spat. Salinity and rearing site also had significant effects on survival, but no significant differences were observed in terms of survival between the diet and diet availability treatments. Growth declined with decrease in salinity. Spat reared at high salinities (30 and 27) showed larger shell length growth and greater survival than those at low salinities (24 and 21). Sp...

Involvement of a novel Ca2+-independent C-type lectin from Sinonovacula constricta in food recognition and innate immunity

Platymonas subcordiformis, a model microalga in microalgal biotechnology, is rich in bioactive substances such as fatty acid, protein and vitamin, and hence it has been widely applied in biofuel, environmental purification, and pharmaceutical industry. In the current study, promotion effects of nitrogen on cell proliferation of P. subcordiformis were investigated by using spectrophotography ana-lysis and cytometry analysis, and the correlation between optical density at wavelength of 680 nm (OD680) and cell density was estimated. The results show that there were significant effects of nitrogen on the growth ofP. subcordiformis, with the OD680, cell density and dry weight increasing with the increase of nitrogen concentration. In the termination of experiment, OD680 values were 0.037, 0.111, 0.188, 0.393, 0.551 and 0.628; cell densities were 4×104, 2.6×105, 4.1×105, 8.3×105, 1.15×106 and 1.38×106 cells/mL; and dry weights were 0.021 7, 0.088 3, 0.131 7, 0.177 5, 0.238 3 and 0.310 0 g/L at the nitrogen concentrations of 0, 7.5, 15, 37.5, 75 and 150 mg/L, respectively. In addition, there was significantly positive correlation between OD680 and cell density throughout the experiment, with the regression equation of cell density (104 cells/mL) = 218.05OD680 - 1.6014 (R2 = 0.997 1) on day 11. The findings suggest that nitrogen promotes the cell growth and proliferation of P. subcordiformis, and that spectrophotography analysis is relatively simple, quick and accurate, which can be used in the scientific research or industrialization of this microalga. Key words: Spectrophotography analysis, cytometry analysis, cell proliferation,Platymonas subcordiformis, nitrogen

Application of Nitrogen and Phosphorus Stress Conditions for Biomass and Oil Production by Chlorella vulgaris Biodiesel from higher plantshas a number of limitations like burden on limited agricultural land and fresh water resources, especially in poor developing nations. On the other hand wastelands can be effectively used for the cultivation of microalgae and potential yields from many green algae have been reported to be many folds to that obtained from the oil crops. Productivity in natural habitats is generally limited by low nitrogen and phosphorus content. Present study deals with the application of main nutrients nitrogen and phosphorus stress conditions commonly found in natural water bodies on stimulating the biofuel components oils in Chlorella vulgaris, a unicellular freshwater green alga and a potential feedstock for biodiesel. Oils from the algal biomass were extracted in chloroform-methanol (2:1) and identified by GC-MS. Significantly reduced concentrations of the nutrients Ca(NO3 )2 and K2 HPO4 from the control values of Chu-10 medium positively affected the oil content without much affecting the growth. There was also a significant effect on the relative ratios of various types of oils produced by the algal strain in response to the nutrient stress conditions. Mostly C14 to C26 fatty acids were identified, with palmitic, oleic and stearic acids being the major ones. The relative proportion of the desirable biofuel component, palmitic acid, was increased significantly under NO3 - and PO4 3- stress conditions. These findings suggest that monitoring the concentrations of a few nutrients may lead to significant enhancement in quantity as well as the desirable biofuel components with substantial growth for commercial cultivation of C. vulgaris for bioenergy production.

Stroma-Based Versus Stroma-Free Long-Term Survival and Proliferation of Primary Acute Myeloid Leukemia (AML) Cells

We investigated the role of extracellular magnesium on capillary endothelial cell migration and proliferation, components of endothelial cell function that play an important role in angiogenesis and wound healing. Cell migration and proliferation were tested in six different MgSO4 concentrations and in various culture conditions. The Boyden chamber procedure was used to evaluate migration of bovine adrenal cortex capillary endothelial cells. We found that low magnesium concentration inhibited cell migration, but a dose-dependent increase in migration was observed when magnesium level was increased beyond the normal serum concentration (up to 2.4 mM magnesium; p less than 0.0001). Cell proliferation was also inhibited by very low magnesium concentration, an effect observed under all conditions studied. When cell proliferation was stimulated by acidic or basic fibroblast growth factors, it appeared that a ceiling was reached, an increasing magnesium concentration had no additional stimulatory effect. However, a dose-dependent increase in proliferation (p less than 0.005) was observed when magnesium concentration was increased above the normal serum level (0.8 mM) in culture conditions that did not cause marked cell proliferation. Thus, magnesium has an important role in endothelial cell migration and proliferation: very low extracellular magnesium concentrations inhibit and supranormal levels enhance both migration and proliferation. These results suggest that magnesium deficiency might adversely influence the healing and reendothelialization of vascular injuries and the healing of myocardial infarction and might also result in delayed or inadequate angiogenesis, effects potentially leading to infarct expansion and inadequate collateral development.

Oxygen tension plays an important role in regulating various cellular functions in both normal physiology and disease states. Therefore, drug testing using conventional in vitro cell models under normoxia often possesses limited prediction capability. A traditional method of setting an oxygen tension in a liquid medium is by saturating it with a gas mixture at the desired level of oxygen, which requires bulky gas cylinders, sophisticated control, and tedious interconnections. Moreover, only a single oxygen tension can be tested at the same time. In this paper, we develop a microfluidic cell culture array platform capable of performing cell culture and drug testing under various oxygen tensions simultaneously. The device is fabricated using an elastomeric material, polydimethylsiloxane (PDMS) and the well-developed multi-layer soft lithography (MSL) technique. The prototype device has 4 × 4 wells, arranged in the same dimensions as a conventional 96-well plate, for cell culture. The oxygen tensions are controlled by spatially confined oxygen scavenging chemical reactions underneath the wells using microfluidics. The platform takes advantage of microfluidic phenomena while exhibiting the combinatorial diversities achieved by microarrays. Importantly, the platform is compatible with existing cell incubators and high-throughput instruments (liquid handling systems and plate readers) for cost-effective setup and straightforward operation. Utilizing the developed platform, we successfully perform drug testing using an anti-cancer drug, triapazamine (TPZ), on adenocarcinomic human alveolar basal epithelial cell line (A549) under three oxygen tensions ranging from 1.4% to normoxia. The developed platform is promising to provide a more meaningful in vitro cell model for various biomedical applications while maintaining desired high throughput capabilities.

Targeting to improve the utilization of lignocellulosic residues in the ethanol processing industry, this work aimed to test if the product inhibition of the enzymatic hydrolysis could be relieved by extractive reaction using aqueous two-phase systems (ATPS). The performance of enzymatic hydrolysis in ATPS is not well defined in literature. In this thesis, this extractive reaction was tested in terms of experimental conversion of sugarcane bagasse, simulations through conceptual process design and economic feasibility. A thermodynamic framework was developed in order to predict ATPS formation. The screening of ATPS and partition coefficient of the solutes were performed in a high throughput station. The ATPS were composed by polymer and salt. The enzymes were represented by the enzymatic cocktail Cellic CTec (Novozymes). The development of this platform consisted of two main parts: determination of phase diagrams (binodal curves and tie lines) and quantification of the solutes (sugar and proteins) in both top and bottom phases. The most promising ATPS were experimentally explored for enzymatic hydrolysis of sugarcane bagasse. Process design simulated two scenarios: hydrolysis occurring in the bottom phase and in the top phase. Topics such as the adsorption of phase forming components to the bagasse fibers and the influence of enzyme load on the hydrolysis were explored. The sugarcane bagasse hydrolysis in ATPS was conceptually assessed through the implementation of a model composed by two parts: hydrolysis and ATPS multi-batch separation. The designed case characterized by the ATPS hydrolysis was compared to the base case defined as conventional hydrolysis. Regarding the thermodynamic modelling of ATPS, the application of Flory-Huggins (FH) model to predict phase separation in polymer-salt systems was assessed. The implementation and analysis of FH theory involved the estimation of interchange energy (푤푖푗) and the calculation of phase diagrams. There were no statistical differences in determining the phase diagram in HTP platforms and bench-scale, verifying the reliability of methods and equipment suggested in this work. Moreover, tailored approaches to quantify the solutes were 8 presented, taking into account the limitations of techniques that can be applied with ATPS due to the interference of phase forming components with the analytics. This fast methodology proposes to screen up to six different polymer-salt systems in eight days and supplies the results to understand the influence of sugar and protein concentrations on their partition coefficients. Exploring experimentally the ATPS hydrolysis provided strategies on how to conduct extractive enzymatic hydrolysis in ATPS and how to explore the experimental results in order to design a feasible process. In the conceptual design of extractive enzymatic hydrolysis, one of the major bottlenecks identified was the partitioning of glucose to both phases. The resultant conceptual process design operates as a tool to evaluate ATPS hydrolysis and compare it to conventional one. On the other hand, the thermodynamic model could not quantitatively describe the data. This occurs mainly because of the strong influence of random experimental errors on the estimation of interchange energy, systematic errors when translating the observed data to calculated partition concentrations, and FH not being an exact description of phase separation in salt based ATPS. The high throughput screening methodology indicated ATPS able to partition sugar and enzymes. The selected ATPS presented no significant improvements to perform the enzymatic conversion of sugarcane bagasse compared to the conventional hydrolysis. The main reasons were the influence of phase forming components on the enzymatic activity and the low selectivity of sugars in the ATPS. To disclose the application of ATPS in the ethanol processing industry, the recovery and reuse of the phase forming components are imperative for economic feasibility. Moreover, the developed high throughput platform could be further employed to exhaustively screen systems to design effective ATPS for the partition of sugars and proteins in polymer-salt systems.

Cell proliferation is a fundamental characteristic of organisms, driven by the holistic functions of multiple proteins encoded in the genome. However, the individual contributions of thousands of genes and the millions of protein molecules they express to cell proliferation are still not fully understood, even in simple eukaryotes. Here, we present a genome-wide translation map of cells during proliferation in the unicellular alga Cyanidioschyzon merolae, based on the sequencing of ribosome-protected messenger RNA fragments. Ribosome profiling has revealed both qualitative and quantitative changes in protein translation for each gene during cell division, driven by the large-scale reallocation of ribosomes. Comparisons of ribosome footprints from non-dividing and dividing cells allowed the identification of proteins involved in cell proliferation. Given that in vivo experiments on two selected candidate proteins identified a division-phase-specific mitochondrial nucleoid protein and a mitochondrial division protein, further analysis of the candidate proteins may offer key insights into the comprehensive mechanism that facilitate cell and organelle proliferation.