Biological Stoichiometric Analysis during Substrate Utilization and Secondary Metabolite Production by Non-Saccharomyces Yeasts Using Grape Pomace Extract as Fermentation Medium

The emerging interest in the search for alternatives to synthetic preservatives has led to various successful research studies exploring the use of yeasts as potential biological control agents and producers of biopreservatives. The findings that yeasts could be used as producers of biopreservatives lacked some engineering considerations regarding cost-effective process design for scale-up, although partial process optimization using renewable agro-waste has been achieved. This study investigated the biological stoichiometry and bioenergetic parameters during yeast growth and secondary metabolites production i.e., biopreservatives from non-Saccharomyces yeasts using grape pomace extract (GPE), a type of agro-waste, as a fermentation medium. This was achieved by reconfirming the optimum production conditions previously found for Candida pyralidae Y1117, Pichia kluyveri Y1125, and Pichia kluyveri Y1164 in GPE broth as a fermentation medium, conditions under which a high amount of yeast cells were obtained. High-density cell cultures were produced, from which the yeast cell pellets were harvested, dried, and combusted for the determination of elemental analysis, heat of combustion, biological stoichiometry, and bioenergetic parameters. This work generated biological stoichiometric models and bioenergetics information that could assist in the design of yeast biochemical conversion system when GPE is used as fermentation medium, thereby, addressing the biochemical engineering aspects that were lacking in a previous biopreservative production study using Candida pyralidae Y1117, Pichia kluyveri Y1125, and Pichia kluyveri Y1164.

Unbiased transcriptomic analysis of chondrocyte differentiation in a high-density cell culture model

ABSTRACTIn the developing vertebrate skeleton, cartilage is an important precursor to the formation of bones. Cartilage is produced by chondrocytes, which derive from embryonic mesoderm and undergo a stereotypical program of differentiation and maturation. Here we modeled this process in vitro, using primary fetal mouse rib chondrocytes in a high-density cell culture model of cartilage differentiation, and performed genome-wide gene expression profiling over the course of culture. The overarching goal of this study was to characterize the molecular pathways involved in cartilage differentiation and maturation. Our results also enable a comprehensive appraisal of distinctions between common in vitro models for cartilage differentiation, and of differences in their molecular resemblance to cartilage formation in vivo.

Chondrogenesis of human amniotic fluid stem cells in Chitosan-Xanthan scaffold for cartilage tissue engineering

Articular chondral lesions, caused either by trauma or chronic cartilage diseases such as osteoarthritis, present very low ability to self-regenerate. Thus, their current management is basically symptomatic, progressing very often to invasive procedures or even arthroplasties. The use of amniotic fluid stem cells (AFSCs), due to their multipotentiality and plasticity, associated with scaffolds, is a promising alternative for the reconstruction of articular cartilage. Therefore, this study aimed to investigate the chondrogenic potential of AFSCs in a micromass system (high-density cell culture) under insulin-like growth factor 1 (IGF-1) stimuli, as well as to look at their potential to differentiate directly when cultured in a porous chitosan-xanthan (CX) scaffold. The experiments were performed with a CD117 positive cell population, with expression of markers (CD117, SSEA-4, Oct-4 and NANOG), selected from AFSCs, after immunomagnetic separation. The cells were cultured in both a micromass system and directly in the scaffold, in the presence of IGF-1. Differentiation to chondrocytes was confirmed by histology and by using immunohistochemistry. The construct cell-scaffold was also analyzed by scanning electron microscopy (SEM). The results demonstrated the chondrogenic potential of AFSCs cultivated directly in CX scaffolds and also in the micromass system. Such findings support and stimulate future studies using these constructs in osteoarthritic animal models.

Application of Acidophilic Microorganism in the Metal Enrichment of Electroplating Sludge Use the Membrane Bioreactor

Background: Bioleaching is an important technology for treating electroplating sludge. Previous researches have focus on improving the leaching rate of metals in electroplating sludge by bioleaching. However, the concentration of heavy metals in the leachate after single leaching was lower, which is quite unfavorable for subsequent metal recovery. Additionally, membrane bioreactors (MBRs) have been widely used in the field of sewage treatment. Research on the application of bioleaching technology combined with MBRs to enrich metals in electroplating sludge has not been reported. Therefore, in this study, we first combined bioleaching technology and MBRs for metal enrichment in electroplating sludge to obtain the key technology of "acid production - electroplating sludge leaching - leachate regeneration -repeated electroplating sludge leaching - achievement of valuable metal enrichment".Results: In this research, through scaling up from the laboratory scale (shake flasks) to a factory-scale application (10 m³ membrane bioreactors), we mastered the key technology of acid production by acidophilic microorganism, and the acid solution can be repeatedly used for metal leaching. The results showed that the MBR maintained high-density cell growth (≈2.1×109/mL) and a stable sulfuric acid production rate (850 L/h) throughout the entire operational period. Under the above conditions, the maximum cycle number (10 times) for enrichment of the target metals in the electroplating sludge was obtained. Additionally, after the end of the cycle enrichment process, the concentrations of the target metals Ni+, Cu2+, and Zn2+ were 13.867 g/L, 18.118 g/L and 21.075 g/L, respectively, which were highly enriched.Conclusions: This study first solved the difficulties in the industrialization of bioleaching electroplating sludge through combining bioleaching technology and MBRs. Furthermore, this research can provide a demonstration project for the industrial application of MBR-bioleaching technology in electroplating sludge, with a view to applying this technology to the disposal of more types of hazardous waste.

Lignocellulosic bioethanol production: prospects of emerging membrane technologies to improve the process – a critical review

To meet the worldwide rapid growth of industrialization and population, the demand for the production of bioethanol as an alternative green biofuel is gaining significant prominence. The bioethanol production process is still considered one of the largest energy-consuming processes and is challenging due to the limited effectiveness of conventional pretreatment processes, saccharification processes, and extreme use of electricity in common fermentation and purification processes. Thus, it became necessary to improve the bioethanol production process through reduced energy requirements. Membrane-based separation technologies have already gained attention due to their reduced energy requirements, investment in lower labor costs, lower space requirements, and wide flexibility in operations. For the selective conversion of biomasses to bioethanol, membrane bioreactors are specifically well suited. Advanced membrane-integrated processes can effectively contribute to different stages of bioethanol production processes, including enzymatic saccharification, concentrating feed solutions for fermentation, improving pretreatment processes, and finally purification processes. Advanced membrane-integrated simultaneous saccharification, filtration, and fermentation strategies consisting of ultrafiltration-based enzyme recycle system with nanofiltration-based high-density cell recycle fermentation system or the combination of high-density cell recycle fermentation system with membrane pervaporation or distillation can definitely contribute to the development of the most efficient and economically sustainable second-generation bioethanol production process.

Robotic Cell Printing for Constructing Living Yeast Cell Microarrays in Microfluidic Chips

Living cell microarrays in microfluidic chips allow the non-invasive multiplexed molecular analysis of single cells. Here, we developed a simple and affordable perfusion microfluidic chip containing a living yeast cell array composed of a population of cell variants (green fluorescent protein (GFP)-tagged Saccharomyces cerevisiae clones). We combined mechanical patterning in 102 microwells and robotic piezoelectric cell dispensing in the microwells to construct the cell arrays. Robotic yeast cell dispensing of a yeast collection from a multiwell plate to the microfluidic chip microwells was optimized. The developed microfluidic chip and procedure were validated by observing the growth of GFP-tagged yeast clones that are linked to the cell cycle by time-lapse fluorescence microscopy over a few generations. The developed microfluidic technology has the potential to be easily upscaled to a high-density cell array allowing us to perform dynamic proteomics and localizomics experiments.