Xingju Yu

A comparative study on the phylogenetic diversity of culturable actinobacteria isolated from five marine sponge species

A cultivation-based approach was employed to compare the culturable actinobacterial diversity associated with five marine sponge species (Craniella australiensis, Halichondria rugosa, Reniochalina sp., Sponge sp., and Stelletta tenuis). The phylogenetic affiliation of the actinobacterial isolates was assessed by 16S rDNA-RFLP analysis. A total of 181 actinobacterial strains were isolated using five different culture media (denoted as M1–M5). The type of medium exhibited significant effects on the number of actinobacteria recovered, with the highest number of isolates on M3 (63 isolates) and the lowest on M1 (12 isolates). The genera isolated were also different, with the recovery of three genera on M2 and M3, and only a single genus on M1. The number of actinobacteria isolated from the five sponge species was significantly different, with a count of 83, 36, 30, 17, and 15 isolates from S. tenuis, H. rugosa, Sponge sp., Reniochalina sp., and C. australiensis, respectively. M3 was the best isolation medium for recovery of actinobacteria from S. tenuis, H. rugosa, and Sponge sp., while no specific medium preference was observed for the recovery of actinobacteria from Reniochalina sp., and C. australiensis. The RFLP fingerprinting of 16S rDNA genes digested with HhaI revealed six different patterns, in which 16 representative 16S rDNAs were fully sequenced. Phylogenetic analysis indicated that 12 strains belong to the group Streptomyces, three strains belong to Pseudonocardia, and one strain belongs to Nocardia. Two strains C14 (from C. australiensis) and N13 (from Sponge sp.) have only 96.26% and 96.27% similarity to earlier published sequences, and are therefore potential candidates for new species. The highest diversity of three actinobacteria genera was obtained from Sponge sp., though the number of isolates was low. Two genera of actinobacteria, Streptomyces, and Pseudonocardia, were isolated from both S. tenuis and C. australiensis. Only the genus of Streptomyces was isolated from H. rugosa and Reniochalina sp. Sponge species have been demonstrated here to vary as sources of culturable actinobacterial diversity, and the methods for sampling such diversity presented may be useful for improved sampling of such diversity.

Optimizing the formation of in vitro sponge primmorphs from the Chinese sponge Stylotella agminata (Ridley).

The establishment and optimization of in vitro primmorph formation from a Chinese sponge, Stylotella agminata (Ridley), collected from the South China Sea, were investigated. Our aims were to identify the key factors affecting primmorph formation in this species and to optimize the technique for developing an in vitro primmorph culture system. The size of dissociated cells from S. agminata is relatively small, in the range between 5 and 10 microm. Round-shaped primmorphs of less than 100 microm were formed 3 days after transferring the dissociated cells into seawater containing Ca(2+) and Mg(2+). The effect of various cell dissociation conditions, inoculum cell density, concentration of antibiotics, pH, and temperature was further investigated upon the formation of primmorphs. The time required for primmorph formation, primmorph size distribution, and the proliferating capability were microscopically documented. Healthy sponge S. agminata, inoculum cell density and culture temperature play a critical role for the successful formation of primmorphs and that the microbial contamination will have to be controlled.

Scalable producing embryoid bodies by rotary cell culture system and constructing engineered cardiac tissue with ES-derived cardiomyocytes in vitro

Embryonic stem (ES) cells are of significant interest either as an in vitro model recapitulating early embryonic development or as a renewable source of therapeutically useful cells. ES cells aggregation is important for embryoid bodies (EBs) formation and the subsequent generation of ES cell derivatives. This study was conducted to describe scalable production of EBs by the rotary cell culture system (RCCS, STLV type) and estimate the feasibility of constructing engineered cardiac tissue (ECT). In comparison with suspension culture in a Petri dish, the efficiency of the dynamic process was analyzed with respect to the yield of EB formation and their cardiomyocyte differentiation. Cardiomyocyte differentiation was evaluated by immunohistochemical analysis. After the elementary enrichment by gradient percoll, ES cell‐derived cardiomyocytes were applied to construct ECT. Cell gross morphology, spatial distribution, and ultrastructure were evaluated by using histological analysis, confocal laser scanning microscopy, and transmission electron microscopy. Results showed that EB efficiencies in STLV were nearly 1.5–2.0 times higher than that of liquid suspension cultures, and cardiomyocyte differentiation of EBs progressed in a normal course after the dynamic cultivation in STLV. Additionally, the differentiated cultures could be enriched elementarily by gradient percoll. Once cast into the collagen strand, cells grew well and became more matured in Petri dishes. Synchronous contraction of the cell cluster was observed on the surface of the ECT, and cell connection was also established. It was the first report to have beating ES‐derived cardiomyocytes on a 3‐D collagen scaffold, which might provide a promising model for physiological and pharmacological studies and tissue replacement therapy.

Role of Carbonyl Cyanide m‐Chlorophenylhydrazone in Enhancing Photobiological Hydrogen Production by Marine Green Alga Platymonas subcordiformis

We demonstrated that a significant volume of H2 gas could be photobiologically produced by a marine green alga Platymonas subcordiformis when an uncoupler of photophosphorylation, carbonyl cyanide m‐chlorophenylhydrazone (CCCP), was added after 32 h of anaerobic dark incubation, whereas a negligible volume of H2 gas was produced without CCCP. The role of CCCP in enhancing photobiological H2 production was delineated. CCCP as an ADRY agent (agent accelerating the deactivation reactions of water‐splitting enzyme system Y) rapidly inhibited the photosystem II (PSII) activity of P. subcordiformis cells, resulting in a markedly decline in the coupled oxygen evolution. The mitochondrial oxidative respiration was only slightly inactivated by CCCP, which depleted O2 in the light. As a result, anaerobiosis during the stage of photobiological H2 evolution was established, preventing severe O2 inactivation of the reversible hydrogenase in P. subcordiformis. The uncoupling effect of CCCP accelerates electron transfer from water due to a disruption of the proton motive force and release of ΔpH across the thylakoid membrane and thus enhances the accessibility of electron and H+ to hydrogenase. The electrons for hydrogen photoevolution are mainly from the photolysis of water (90%). Upon the addition of CCCP, Chl a/b ratio increased, which implies a decrease in the light‐harvesting PSII antennae or an increase in PSII/PSI ratio, possibly resulting in higher efficiency of utilization of light energy. The enhancement of H2 evolution by the addition of CCCP is mostly due to the combination of the above three mechanisms. However, the disruption of the proton gradient across the thylakoid membrane may prevent a sustained photobiological H2 evolution due to a shortfall of ATP generation essential for the maintenance and repair functions of the cells.

Formulation of a basal medium for primary cell culture of the marine sponge Hymeniacidon perleve.

Marine sponge cell culture is a potential route for the sustainable production of sponge‐derived bioproducts. Development of a basal culture medium is a prerequisite for the attachment, spreading, and growth of sponge cells in vitro. With the limited knowledge available on nutrient requirements for sponge cells, a series of statistical experimental designs has been employed to screen and optimize the critical nutrient components including inorganic salts (ferric ion, zinc ion, silicate, and NaCl), amino acids (glycine, glutamine, and aspartic acid), sugars (glucose, sorbitol, and sodium pyruvate), vitamin C, and mammalian cell medium (DMEM and RPMI 1640) using MTT assay in 96‐well plates. The marine sponge Hymeniacidon perleve was used as a model system. Plackett‐Burman design was used for the initial screening, which identified the significant factors of ferric ion, NaCl, and vitamin C. These three factors were selected for further optimization by Uniform Design and Response Surface Methodology (RSM), respectively. A basal medium was finally established, which supported an over 100% increase in viability of sponge cells.

Instability of Anthocyanin Accumulation in Vitis vinifera L. var. Gamay Freaux Suspension Cultures

The inherent instability of metabolite production in plant cell culture-based bioprocessing is a major problem hindering its commercialization. To understand the extent and causes of this instability, this study was aimed at understanding the variability of anthocyanin accumulation during long-term subcultures, as well as within subculture batches, inVitis vinifera cell cultures. Therefore, four cell line suspensions ofVitis vinifera L. var. Gamay Fréaux, A, B, C and D, originated from the same callus by cell-aggregate cloning, were established with starting anthocyanin contents of 2.73±0.15, 1.45±0.04, 0.77±0.024 and 0.27±0.04 CV (Color Value)/g-FCW (fresh cell weight), respectively. During weekly subculturing of 33 batches over 8 months, the anthocyanin biosynthetic capacity was gradually lost at various rates, for all four cell lines, regardless of the significant difference in the starting anthocyanin content. Contrary to this general trend, a significant fluctuation in the anthocyanin content was observed, but with an irregular cyclic pattern. The variabilities in the anthocyanin content between the subcultures for the 33 batches, as represented by the variation coefficient (VC), were 58, 57, 54, and 84% forV. vinifera cell lines A, B, C and D, respectively. Within one subculture, the VCs from 12 replicate flasks for each of 12 independent subcultures were averaged, and found to be 9.7%, ranging from 4 to 17%. High- and low-producing cell lines, VV05 and VV06, with 1.8-fold differences in their basal anthocyanin contents, exhibited different inducibilities tol-phenylalanine feeding, methyl jasmonate and light irradiation. The low-producing cell line showed greater potential in enhanced the anthocyanin production.

Attachment of marine sponge cells of Hymeniacidon perleve on microcarriers

Toward the development of an in vitro cultivation of marine sponge cells for sustainable production of bioactive metabolites, the attachment characteristics of marine sponge cells of Hymeniacidon perleve on three types of microcarriers, Hillex, Cytodex 3, and glass beads, were studied. Mixed cell population and enriched cell fractions of specific cell types by Ficoll gradient centrifugation (6%/8%/15%/20%) were also assessed. Cell attachment ratio (defined as the ratio of cells attached on microcarrier to the total number of cells in the culture) on glass beads is much higher than that on Cytodex 3 and Hillex for both mixed cell population and cell fraction at Ficoll 15–20% interface. The highest attachment ratio of 41% was obtained for the cell fraction at Ficoll 15–20% interface on glass beads, which was significantly higher than that of a mixed cell population (18%). The attachment kinetics on glass beads indicated that the attachment was completed within 1 h. Cell attachment ratio decreases with increase in cell‐to‐microcarrier ratio (3–30 cells/bead) and pH (7.6–9.0). The addition of serum and BSA (bovine serum albumin) reduced the cell attachment on glass beads.

Comparison of spiculogenesis in in vitro ADCP-primmorph and explants culture of marine sponge Hymeniacidon perleve with 3-TMOSPU supplementation

This study aims to test the feasibility of introducing functional chemical groups into biogenic silica spicules by examining the effect of supplementing a silican coupler [3‐(trimethoxysilyl)propyl]urea (3‐TMOSPU) as silica source in the cultures of archaeocytes‐dominant‐cell‐population (ADCP) primmorphs and explants of the marine sponge Hymeniacidon perleve. Analysis by Fourier Transform Infrared Spectroscopy (FT‐IR) confirmed that the organic group in 3‐TMOSPU was introduced into silica spicules. By comparing ADCP‐primmorph cultures when supplemented with Na2SiO3, 3‐TMOSPU supplementation showed no notable effect on the primmorphs development and cell locomotion behaviors. A decline in silicatein expression quantified by real‐time RT‐PCR was, however, observed during spiculogenesis. The decline was slower for the 3‐TMOSPU group whereas significantly fewer spicules were formed. When sponge papillae explants were cultured, 3‐TMOSPU supplementation had no negative effect on sponge growth but inhibited the growth biofouling of the diatom Nitzschia closterium. By monitoring the detectable Si concentration, it seemed that 3‐TMOSPU was converted by the sponge and its conversion was related to spiculogenesis. Analysis of spicule dimensional changes indicated that the inhibition of spiculogenesis by 3‐TMOSPU supplementation was less in ADCP‐primmorphs culture due to lower 3‐TMOSPU/detectable Si ratio in the media.