On-Line Monitoring of Biological Parameters in Microalgal Bioprocesses Using Optical Methods

Microalgae are promising sources of fuels and other chemicals. To operate microalgal cultivations efficiently, process control based on monitoring of process variables is needed. On-line sensing has important advantages over off-line and other analytical and sensing methods in minimizing the measurement delay. Consequently, on-line, in-situ sensors are preferred. In this respect, optical sensors occupy a central position since they are versatile and readily implemented in an on-line format. In biotechnological processes, measurements are performed in three phases (gaseous, liquid and solid (biomass)), and monitored process variables can be classified as physical, chemical and biological. On-line sensing technologies that rely on standard industrial sensors employed in chemical processes are already well-established for monitoring the physical and chemical environment of an algal cultivation. In contrast, on-line sensors for the process variables of the biological phase, whether biomass, intracellular or extracellular products, or the physiological state of living cells, are at an earlier developmental stage and are the focus of this review. On-line monitoring of biological process variables is much more difficult and sometimes impossible and must rely on indirect measurement and extensive data processing. In contrast to other recent reviews, this review concentrates on current methods and technologies for monitoring of biological parameters in microalgal cultivations that are suitable for the on-line and in-situ implementation. These parameters include cell concentration, chlorophyll content, irradiance, and lipid and pigment concentration and are measured using NMR, IR spectrophotometry, dielectric scattering, and multispectral methods. An important part of the review is the computer-aided monitoring of microalgal cultivations in the form of software sensors, the use of multi-parameter measurements in mathematical process models, fuzzy logic and artificial neural networks. In the future, software sensors will play an increasing role in the real-time estimation of biological variables because of their flexibility and extendibility

Variation in honey bee gut microbial diversity affected by ontogenetic stage, age and geographic location

Social honey bees, Apis mellifera, host a set of distinct microbiota, which is similar across the continents and various honey bee species. Some of these bacteria, such as lactobacilli, have been linked to immunity and defence against pathogens. Pathogen defence is crucial, particularly in larval stages, as many pathogens affect the brood. However, information on larval microbiota is conflicting. Seven developmental stages and drones were sampled from 3 colonies at each of the 4 geographic locations of A. mellifera carnica, and the samples were maintained separately for analysis. We analysed the variation and abundance of important bacterial groups and taxa in the collected bees. Major bacterial groups were evaluated over the entire life of honey bee individuals, where digestive tracts of same aged bees were sampled in the course of time. The results showed that the microbial tract of 6-day-old 5th instar larvae were nearly equally rich in total microbial counts per total digestive tract weight as foraging bees, showing a high percentage of various lactobacilli (Firmicutes) and Gilliamella apicola (Gammaproteobacteria 1). However, during pupation, microbial counts were significantly reduced but recovered quickly by 6 days post-emergence. Between emergence and day 6, imago reached the highest counts of Firmicutes and Gammaproteobacteria, which then gradually declined with bee age. Redundancy analysis conducted using denaturing gradient gel electrophoresis identified bacterial species that were characteristic of each developmental stage. The results suggest that 3-day 4th instar larvae contain low microbial counts that increase 2-fold by day 6 and then decrease during pupation. Microbial succession of the imago begins soon after emergence. We found that bacterial counts do not show only yearly cycles within a colony, but vary on the individual level. Sampling and pooling adult bees or 6th day larvae may lead to high errors and variability, as both of these stages may be undergoing dynamic succession

Monitoring of microalgal cultivations with on-line, flow-through microscopy

Microalgal cultivations present challenges for monitoring and process control posed by their large scale and the likelihood that they will be composed of multiple species. Cell concentration is a fundamental parameter in any cultivation but is typically measured using off-line methods that may be time-consuming, laborious, or subject to interferences. Here, an in-situ microscope has been adapted for monitoring microalgal cultivations by adding a flow-through cell and adjusting image-processing algorithms. After installation in the bypass of a photobioreactor, the microscope enabled the continuous, automated acquisition of cell count, cell size, and cell morphology data on-line during cultivation processes over a period of 20. days, without sampling. The flow-through microscope was tested in cultivations of Chlamydomonas reinhardtii and Chlorella vulgaris. Cell concentration measurements were in agreement with off-line optical density measurements for both species. In addition, cell size and morphology distributions were obtained that revealed population shifts during the cultivation of C. vulgaris. This monitoring system thus provides a means to obtain detailed, non-invasive insights of microalgal cultivation processes.Jud and Pat Harper Professorship in Chemical and Biological EngineeringSustainable Bioenergy Development Center of Colorado State Universit

New Broth Macrodilution Volatilization Method for Antibacterial Susceptibility Testing of Volatile Agents and Evaluation of Their Toxicity Using Modified MTT Assay In Vitro

In this study, a new broth macrodilution volatilization method for the simple and rapid determination of the antibacterial effect of volatile agents simultaneously in the liquid and vapor phase was designed with the aim to assess their therapeutic potential for the development of new inhalation preparations. The antibacterial activity of plant volatiles (β-thujaplicin, thymohydroquinone, thymoquinone) was evaluated against bacteria associated with respiratory infections (Haemophilus influenzae, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes) and their cytotoxicity was determined using a modified thiazolyl blue tetrazolium bromide assay against normal lung fibroblasts. Thymohydroquinone and thymoquinone possessed the highest antibacterial activity against H. influenzae, with minimum inhibitory concentrations of 4 and 8 µg/mL in the liquid and vapor phases, respectively. Although all compounds exhibited cytotoxic effects on lung cells, therapeutic indices (TIs) suggested their potential use in the treatment of respiratory infections, which was especially evident for thymohydroquinone (TI > 34.13). The results demonstrate the applicability of the broth macrodilution volatilization assay, which combines the principles of broth microdilution volatilization and standard broth macrodilution methods. This assay enables rapid, simple, cost- and labor-effective screening of volatile compounds and overcomes the limitations of assays currently used for screening of antimicrobial activity in the vapor phase

Metabolism of Selected 2-Arylbenzofurans in a Colon In Vitro Model System

2-arylbenzofurans represent a small group of bioactive compounds found in the plant family Moraceae. As it has not been investigated whether these substances are stable during passage through the gastrointestinal tract, their biological effects may be altered by the metabolism of intestinal microbiota or cells. The aim of the present study was to investigate and compare mulberrofuran Y (1), moracin C (2), and mulberrofuran G (3) in an in vitro model of human intestinal bacterial fermentation and in an epithelial model using the Caco-2 cell line. The analysis of compounds by LC-MS-Q-TOF showed sufficient stability in the fermentation model, with no bacterial metabolites detected. However, great differences in the quantity of permeation were observed in the permeability assay. Moreover, mulberrofuran Y (1) and moracin C (2) were observed to be transformed into polar metabolites by conjugation. Among the test compounds, mulberrofuran Y (1) was mostly stable and accumulated in endothelial cells (85.3%) compared with mulberrofuran G (3) and moracin C (2) (14% and 8.2%, respectively). Thus, only a small amount of mulberrofuran Y (1) was conjugated. Moracin C (2) and mulberrofuran G (3) were metabolized almost completely, with only traces of the unchanged molecule being found on the apical and cellular sides of the system. Only conjugates of mulberrofuran Y (1) and moracin C (2) were able to reach the basolateral side. Our results provide the basic description of bioavailability of these three compounds, which is a necessary characteristic for final evaluation of bio-efficacy

In vitro antioxidant and anti-proliferative activity of Ethiopian medicinal plant extracts

Identification and characterization of natural products with antioxidant and anti-proliferative activity has received much interest over the past few years. Ethiopia is one of the developing countries which have enormous diversity of plants and yet majority stays scientifically neglected and undiscovered. In this study, the ethanol extracts of 18 Ethiopian wild medicinal plants were investigated for their in vitro antioxidant and anti-proliferative potential. For this purpose DPPH (2,2-diphenyl-1-picrylhydrazyl), ORAC (oxygen radical absorbance capacity) and TPC (total phenolic content) assays together with MTT cell viability assay (performed on Hep-G2 and MRC-5) were used. Extracts of Carissa spinarum, Dodonaea angustifolia, Jasminum abyssinicum, Rumex nepalensis, Rubus steudneri and Verbascum sinaiticum exhibited the most significant results. However, it was discovered that C. spinarum, J. abyssinicum and R. steudneri possessed considerable toxicity against normal MRC-5 cell line. Only extracts of D. angustifolia and R. nepalensis demonstrated significant combinatory antioxidant/anti-proliferative effect, while V. sinaiticum showed best selective anti-proliferative activity. Since aforementioned extracts also exerted low or minimal toxicity to normal cells, we suggest these as prospective material for further development of novel plant-based agents effective against oxidative stress related diseases

In vitro immunomodulatory activity, cytotoxicity and chemistry of some central European polypores

Context: Some mushrooms of the order Polyporales are known for their immunomodulatory actions. Objective: The objective of this study is to evaluate the in vitro phagocytic and cytotoxic effects of extracts from polyporales native to Central Europe. Materials and methods: The effects of ethanol extracts from 27 polypore species on opsonized zymosan-induced phagocytosis of isolated human neutrophils were tested by a chemiluminescence method. Colon epithelial cell lines, Caco-2 and HT-29, were used for cytotoxicity assays, and extracts were chemically characterized in terms of total phenolic and β-glucan content. Results: We observed phagocytosis or respiratory burst enhancing activity in 17 extracts, of which five species, namely Aurantiporus fissilis (Berk. & M.A. Curtis) H. Jahn ex Ryvarden, Trametes gibbosa (Pers.) Fr., Piptoporus betulinus (Bull.) P. Karst, Neolentinus lepideus (Fr.) Redhead & Ginns, Polyporus squamosus (Huds.) Fr., significantly increased phagocytosis in granulocytes by 205, 181, 158, 155 and 141%, respectively. The β-glucan content of the three most potent extracts was 58, 42 and 74 mg/g, respectively, and the polyphenol content was 155.6, 133.5 and 155.2 μmol of gallic acid equivalent/g, respectively. Some extracts showed cytotoxic activity, with higher cytotoxicity in Caco-2 than in HT-29 cells. Pycnoporus cinnabarinus (Jacq.) P. Karst. extract was cytotoxic to both cell lines, with IC50 values of 81 and 31 μg/mL, respectively. Discussion and conclusion: The most promising extracts were from N. lepideus and Polyporus squamosus, which are edible species and may be considered safe. Our findings support their use as culinary preparations or food supplements for various immunological gut disorders

Cytotoxic activities of Amaryllidaceae alkaloids against gastrointestinal cancer cells

The treatment of many diseases is highly dependent on natural products and natural products can also be used as design templates for future anticancer drugs. Thirteen Amaryllidaceae alkaloids possessing α-crinane, β-crinane, galantamine, lycorine and tazettine-type skeleton have been isolated in our laboratory, and their cytotoxicity against p53-mutated gastrointestinal cancer cells were evaluated. At the same time, healthy small intestine cells were used to determine overall toxicity against noncancerous cells. In this study, we demonstrated that haemanthamine, haemanthidine and lycorine showed strong cytotoxicity against p53-mutated Caco-2 and HT-29 colorectal adenocarcinoma cells as quantified in terms of IC50 values. We for the first time observed approximately 20 times higher IC50values against normal intestine epithelial cells FHs-74 Int after haemanthamine and lycorine treatment when compared with Caco-2 and HT-29 cancer cells. In conclusion, our data indicate that α-C2 bridged haemanthamine may be perspective anticancer drug candidate for further semisynthetic modification and structure-activity relationship study

Phenolic composition, antioxidant and anti-proliferative activities of edible and medicinal plants from the Peruvian Amazon

Among 23 extracts of medicinal and edible plants tested, Mauritia flexuosa L.f., Arecaceae, showed significant antioxidant ability (DPPH and ORAC = 1062.9 and 645.9 ± 51.4 μg TE/mg extract, respectively), while Annona montana Macfad., Annonaceae, demonstrated the most promising anti-proliferative effect (IC50 for Hep-G2 and HT-29 = 2.7 and 9.0 μg/ml, respectively). However, combinatory antioxidant/anti-proliferative effect was only detected in Oenocarpus bataua Mart., Arecaceae (DPPH = 903.8 and ORAC = 1024 μg TE/mg extract; IC50 for Hep-G2 and HT-29 at 102.6 and 38.8 μg/ml, respectively) and Inga edulis Mart., Fabaceae (DPPH = 337.0 and ORAC = 795.7 μg TE/mg extract; IC50 for Hep-G2 and HT-29 at 36.3 and 57.9 μg/ml, respectively). Phenolic content was positively correlated with antioxidant potential, however not with anti-proliferative effect. None of these extracts possessed toxicity towards normal foetal lung cells, suggesting their possible use in development of novel plant-based agents with preventive and/or therapeutic action against oxidative stress-related diseases. Keywords: Antioxidant, Anticarcinogenic, Phenolic compounds, Plant extract