Optimization of Light Intensity and NaNO3 Concentration in Amazon Cyanobacteria Cultivation to Produce Biodiesel.

The objective of this study, for the first time, was to optimize Amazonian cyanobacterial culture conditions for improving cell productivity and lipid content, by analyzing the effect of light intensity and nitrogen concentration, for empirically evaluating biodiesel quality parameters. The strains Synechocystis sp. CACIAM05, Microcystis aeruginosa CACIAM08, Pantanalinema rosaneae CACIAM18, and Limnothrix sp. CACIAM25, were previously identified by morphological and molecular analysis (16S rRNA) and were selected based on their production of chlorophyll a and dry cell weight. Then, factorial planning (22) with central points was applied, with light intensity and NaNO3 concentration as independent variables. As response variables, cell productivity and lipid content were determined. Statistical analysis indicated that for all strains, the independent variables were statistically significant for cell productivity. Analysis of the fatty acid composition demonstrated diversity in the composition of the fatty acid profile from the experimental planning assays of each strain. The Biodiesel Analyzer software predicted the biodiesel quality parameters. CACIAM05 and CACIAM25 obtained better parameters with low levels of light intensity and NaNO3 concentration, whereas CACIAM08 and CACIAM18 obtained better parameters with low NaNO3 concentrations and high luminous intensity.

Integrated bioprocessing and genetic strategies to enhance soluble expression of anti-HER2 immunotoxin in E. Coli.

Immunotoxins are widely applied for cancer therapy. However, bacterial expression of immunotoxins usually leads to the formation of insoluble and non-functional recombinant proteins. This study was aimed to improve soluble expression of a novel anti-HER2 immunotoxin under the regulation of the trc promoter in Escherichia coli by optimization of the cultivation conditions using response surface methodology (RSM). To conduct RSM, four cultivation variables (i.e., inducer concentration, post-induction time, post-induction temperature, and medium recipe), were selected for statistical characterization and optimization using the Box-Behnken design and Design Expert software. Based on the developed model using the Box-Behnken design, the optimal cultivation conditions for soluble expression of anti-HER2 immunotoxin were determined to be 0.1 mM IPTG for induction in the LB medium at 33 °C for 18 h. The expressed immunotoxin was successfully purified using affinity chromatography with more than 90% purity and its bioactivity was confirmed using cell-based ELISA. Technical approach developed in this study can be generally applied to enhance the production yield and quality of recombinant proteins using E. coli as the gene expression system.

Comparative transcriptomic insights into the domestication of Pleurotus abieticola for coniferous cultivation.

Introduction: Pleurotus abieticola, a promising edible fungus in the Pleurotaceae family, especially its ability to utilize coniferous substrate, holds significant potential for commercial cultivation. However, few reports on the adaptation of P. abieticola to coniferous substrate from the perspective of omics. Methods: This study explores the biological characteristics, domestication process, and nutritional composition of P. abieticola, along with its adaptability to coniferous substrates using transcriptomics. We assessed biological characteristics, optimizing mycelial growth on agar medium with varied carbon and nitrogen sources, temperature, and pH. Additionally, the optimization process extended to fruiting bodies, where impact on the differentiation were evaluated under varying light conditions. Fruiting body nutrient composition was analyzed per the Chinese National Food Safety Standard. Transcriptome sequencing focused on P. abieticola mycelial colonized coniferous and broadleaved substrates. Results and Discussion: The optimal conditions for mycelial growth were identified: dextrin (carbon source), diammonium hydrogen phosphate (nitrogen source), 25°C (temperature), and pH 7.0. White light promoted fruiting body growth and differentiation. Larch substrate exhibited superior yield (190 g) and biological efficiency (38.0%) compared to oak (131 g, 26.2%) and spruce (166 g, 33.2%). P. abieticola showcased high dietary fiber, protein, and total sugar content, low fat, and sufficient microelements. Transcriptome analysis revealed significant key genes involved in lignocellulose degradation, stress-resistant metabolism, and endocytosis metabolism, underscoring their pivotal for coniferous adaptation. This study offers valuable insights for the commercial development and strain breeding of P. abieticola, efficiently leveraging conifer resources. The findings underscore its potential as a valuable source for food, medicinal products, and biotechnological applications.

Increased Rate of Yeast Cultivation from Packaged Beer with Environmentally Relevant Anaerobic Handling.

Beer production necessitates oxygen exclusion for the proper packaging and aging of the beer. Standard operating procedures, including those for quality testing, involve culturing microbes from packaged beer exposed to atmospheric oxygen, despite the generalized fact that packaged beer is an anaerobic environment. Our research goal was to apply an environmentally relevant culturing approach to improve yeast cultivation from bottled beer by attempting to ameliorate transplant shock. This is applicable to uniquely scrutinous quality assurance/control objectives and/or to grand cultivation goals, such as ancient beer samples. Although yeasts have the genetic capacity of oxygen protection, their epigenetic/biochemical states within anaerobic packaging may not adequately protect all cells from reactive oxygen species (ROS) at the moment of opening. Soon after opening, beer yeasts were found to be catalase negative, indicating deficient protection from at least one ROS. The general reduction/inhibition of growth was observed when the beer yeast was exposed to ROS in media, and atmospheric bottle opening was found to expose beer yeast to significantly increased levels of ROS. Our primary finding is that different oxygen handling methodologies (aerobic/microaerophilic/anaerobic) significantly impact the viable Saccharomyces yeast recovery rates of Bamberger's Mahr's Bräu Unfiltered Lager. Immediate anaerobic handling improved cultivation success rates, with significantly higher colony forming units (CFU)/mL being cultured, and reduced the volume of beer required to recover viable yeast. Aerobic standard operating procedures have mainly been developed to harvest yeast on large volumetric samples and/or samples with high viable cell numbers, but these procedures may be suboptimal and may underrepresent potential viable cell numbers. IMPORTANCE Procedures of beer production and packaging exclude oxygen to create a shelf-stable anaerobic environment, within which any viable organisms are stored. However, standard methodologies to cultivate microbes from such environments generally include opening in an oxygenated atmosphere. This study applies environmentally relevant culturing methods and compares the yeast recovery rates of beers handled in various oxygen conditions. When beer bottles were opened in anoxic conditions, higher colony counts were obtained, so a smaller volume of beer was required to recover viable cells. The yeast in beer, stored anaerobically, may not be biochemically prepared to fully protect cells from oxygen at the moment of opening. Negative catalase activity showed beer yeasts' vulnerabilities to reactive oxygen. Atmospheric opening may reduce viability, causing the underreporting of viable cells. Anaerobic opening could increase the odds of successfully detecting/cultivating viable cell(s) that are present, which is pertinent to uniquely stringent quality screens and ambitious culturing attempts from rare samples.

Multispectral Imaging Flow Cytometry with Spatially and Spectrally Resolving Snapshot-Mosaic Cameras for the Characterization and Classification of Bioparticles.

In the development and optimization of biotechnological cultivation processes the continuous monitoring through the acquisition and interpretation of spectral and morphological properties of bioparticles are challenging. There is therefore a need for the parallel acquisition and interpretation of spatially and spectrally resolved measurements with which particles can be characterized and classified in-flow with high throughput. Therefore, in this paper we investigated the scientific and technological connectivity of standard imaging flow cytometry (IFC) with filter-on-chip based spatially and spectrally resolving snapshot-mosaic cameras for photonic sensing and control in a smart and innovative microfluidic device. For the investigations presented here we used the microalgae Haematococcus pluvialis (HP). These microalgae are used commercially to produce the antioxidant keto-carotenoid astaxanthin. Therefore, HP is relevant to practically demonstrate the usability of the developed system for Multispectral Imaging Flow Cytometry (MIFC) platform. The extension of standard IFC with snapshot-mosaic cameras and multivariate data processing is an innovative approach for the in-flow characterization and derived classification of bioparticles. Finally, the multispectral data acquisition and the therefore developed methodology is generalizable and enables further applications far beyond the here characterized population of HP cells.

Kinetic modeling and process analysis for photo-production of ß-carotene in Dunaliella salina.

Dunaliella salina is a green microalga with the great potential to generate natural ß-carotene. However, the corresponding mathematical models to guide optimized production of ß-carotene in Dunaliella salina (D. salina) are not yet available. In this study, dynamic models were proposed to simulate effects of environmental factors on cell growth and ß-carotene production in D. salina using online monitoring system. Moreover, the identification model of the parameter variables was established, and an adaptive particle swarm optimization algorithm based on parameter sensitivity analysis was constructed to solve the premature problem of particle swarm algorithm. The proposed kinetic model is characterized by high accuracy and predictability through experimental verification, which indicates its competence for future process design, control, and optimization. Based on the model established in this study, the optimal environmental factors for both ß-carotene production and microalgae growth were identified. The approaches created are potentially useful for microalga Dunaliella salina cultivation and high-value ß-carotene production.

Multi-factor kinetic modelling of microalgal biomass cultivation for optimised lipid production.

This paper presents a new quadruple-factor kinetic model of microalgal cultivation considering carbon and nitrogen concentration, light intensity and temperature, developed in conjunction with laboratory-scale experiments using the well-studied chlorophyte microalgal species Chlamydomonas reinhardtii. Multi-parameter quantification was exploited to assess the predictive capabilities of the model. The validated model was utilized in an optimization study to determine the optimal light intensity and temperature for achieving maximum lipid productivity while using optimal acetate and nitrogen concentrations (2.1906 g L-1 acetate and 0.0742 g L-1 nitrogen) computed in a recent publication. It was found that the optimal lipid productivity increased by 50.9% compared to the base case, and by 13.6% compared to the previously computed optimal case. Optimization results were successfully validated experimentally. Such comprehensive modelling approaches can be exploited for robust design, scale-up and optimization of microalgal oil production, reducing operating costs and bringing this important technology closer to industrialization.

Heterotrophy of filamentous oleaginous microalgae Tribonema minus for potential production of lipid and palmitoleic acid.

Heterotrophic fermentation and high valuable co-product producing are thought to be effective ways to improve the economic viability and feasibility of commercial production of microalgae biofuels. This work reported the heterotrophic cultivation of Tribonema minus for lipid and palmitoleic acid (a novel functional fatty acid) production. Firstly, the heterotrophic ability of T. minus was identified for the first time with significant promotion in biomass and lipid productivity, and glucose and urea were then selected as the optimal carbon and nitrogen sources. Moreover, nutrient concentrations and culture conditions were optimized. Highest biomass and lipid productivity of 30.8gL-1 and 730mgL-1d-1 were obtained respectively by adding 80gL-1 glucose at once. In addition, 2gL-1 urea, 0.8gL-1 K2HPO4, 24mgL-1 ammonium ferric citrate, initial pH of 6, and temperature of 27°C were determined as the appropriate conditions for heterotrophic growth and lipid production.

Fluorescence based cell counting in collagen monolayer cultures of primary hepatocytes.

Accurate determination of cell number is essential for the quantitative description of biological processes. The changes should be related to a measurable reference e.g. in the case of cell culture, the viable cell number is a very valuable reference parameter. Indirect methods of cell number/viability measurements may have up to 10 % standard deviation. This can lead to undesirable large deviations in the analysis of "-omics" data as well as time course studies. Such data should be preferably normalized to the exact viable cell number at a given time to allow meaningful interpretation and understanding of the biological processes. Manual counting of cell number is very laborious and not possible in certain experimental setups. We therefore, developed a simple and reliable fluorescence based method with an accuracy of 95-98 % for the determination of the viable cell number in situ. We optimized the seeding cell densities for primary rat hepatocytes for optimal cell adhesion. This will help in efficient use of primary cells which are usually limited in availability. The method will be very useful in the application of "-omics" techniques, especially metabolome analysis where the specific rates of uptake/production of metabolites can be reliably calculated.