Batch Culture Conditions Research Articles

Strain-specific morphological response of the dominant calcifying phytoplankton species Emiliania huxleyi to salinity change.

The future physiology of marine phytoplankton will be impacted by a range of changes in global ocean conditions, including salinity regimes that vary spatially and on a range of short- to geological timescales. Coccolithophores have global ecological and biogeochemical significance as the most important calcifying marine phytoplankton group. Previous research has shown that the morphology of their exoskeletal calcified plates (coccoliths) responds to changing salinity in the most abundant coccolithophore species, Emiliania huxleyi. However, the extent to which these responses may be strain-specific is not well established. Here we investigated the growth response of six strains of E. huxleyi under low (ca. 25) and high (ca. 45) salinity batch culture conditions and found substantial variability in the magnitude and direction of response to salinity change across strains. Growth rates declined under low and high salinity conditions in four of the six strains but increased under both low and high salinity in strain RCC1232 and were higher under low salinity and lower under high salinity in strain PLYB11. When detailed changes in coccolith and coccosphere size were quantified in two of these strains that were isolated from contrasting salinity regimes (coastal Norwegian low salinity of ca. 30 and Mediterranean high salinity of ca. 37), the Norwegian strain showed an average 26% larger mean coccolith size at high salinities compared to low salinities. In contrast, coccolith size in the Mediterranean strain showed a smaller size trend (11% increase) but severely impeded coccolith formation in the low salinity treatment. Coccosphere size similarly increased with salinity in the Norwegian strain but this trend was not observed in the Mediterranean strain. Coccolith size changes with salinity compiled for other strains also show variability, strongly suggesting that the effect of salinity change on coccolithophore morphology is likely to be strain specific. We propose that physiological adaptation to local conditions, in particular strategies for plasticity under stress, has an important role in determining ecotype responses to salinity.

Purification, Characterization, Optimization and Evaluation of Bioactivity Potential of Exopolysaccharides of Curvularia Lunata

Extracellular polymeric substances are present abundantly in the surroundings which are produced by different types of microorganisms like bacteria, fungi, algae are also produced by different varieties of plant species. Exopolysaccharides (EPS) are high-molecular-weight polysaccharides secreted by microorganisms into the surrounding environment. These polysaccharides have tremendous application in pharmaceutical industries. For the past two decades, exopolysaccharides of fungus have been known to have high-value biomacromolecules. In most of the studies, sugar compositions of fungus Exopolysaccharaide and variation in molecular weight depend upon culture medium composition and different physical conditions that take place during the process of fermentation. In this current study, the selection of nutrients for the EPS production from Curvularia lunata under submerged batch-culture conditions was made using the Plackett - Burman design matrix method. Subsequently, the optimal condition was determined using Response Surface Methodology (RSM). Thus, produced EPS was characterized by GC-MS and thermogravimetric analysis. It was found to have glucose and mannitol and also showed good thermal stability. EPS was subjected to DPPH assay for analyzing antioxidant activity. Free radical scavenging activity was observed by subjecting the sample to the ABTS method and anticancer activities were tested against HeLa cells. EPS was found to be having a good antioxidant activity, Free radical scavenging activity and showed effective anticancer activity against HeLa cells.

Effects of Nitrogen Availability on the Antioxidant Activity and Carotenoid Content of the Microalgae Nephroselmis sp.

Nephroselmis sp. was previously identified as a species of interest for its antioxidant properties owing to its high carotenoid content. In addition, nitrogen availability can impact biomass and specific metabolites’ production of microalgae. To optimize parameters of antioxidant production, Nephroselmis sp. was cultivated in batch and continuous culture conditions in stirred closed photobioreactors under different nitrogen conditions (N-repletion, N-limitation, and N-starvation). The aim was to determine the influence of nitrogen availability on the peroxyl radical scavenging activity (oxygen radical absorbance capacity (ORAC) assay) and carotenoid content of Nephroselmis sp. Pigment analysis revealed a specific and unusual photosynthetic system with siphonaxanthin-type light harvesting complexes found in primitive green algae, but also high lutein content and xanthophyll cycle pigments (i.e., violaxanthin, antheraxanthin, and zeaxanthin), as observed in most advanced chlorophytes. The results indicated that N-replete conditions enhance carotenoid biosynthesis, which would correspond to a higher antioxidant capacity measured in Nephroselmis sp. Indeed, peroxyl radical scavenging activity and total carotenoids were higher under N-replete conditions and decreased sharply under N-limitation or starvation conditions. Considering individual carotenoids, siphonaxanthin, neoxanthin, xanthophyll cycle pigments, and lycopene followed the same trend as total carotenoids, while β-carotene and lutein stayed stable regardless of the nitrogen availability. Carotenoid productivities were also higher under N-replete treatment. The peroxyl radical scavenging activity measured with ORAC assay (63.6 to 154.9 µmol TE g−1 DW) and the lutein content (5.22 to 7.97 mg g−1 DW) were within the upper ranges of values reported previously for other microalgae. Furthermore, contents of siphonaxanthin ere 6 to 20% higher than in previous identified sources (siphonous green algae). These results highlight the potential of Nephroselmis sp. as a source of natural antioxidant and as a pigment of interest.

Physiological and transcriptomic responses to N-deficiency and ammonium: Nitrate shift in Fugacium kawagutii (Symbiodiniaceae)

Symbiodiniaceae are the source of essential coral symbionts of reef building corals. The growth and density of endosymbiotic Symbiodiniaceae within the coral host is dependent on nutrient availability, yet little is known about how Symbiodiniaceae respond to the dynamics of the nutrients, including switch between different chemical forms and changes in abundance. In this study, we investigated physiological, cytometric, and transcriptomic responses in Fugacium kawagutii to nitrogen (N)-nutrient deficiency and different chemical N forms (nitrate and ammonium) in batch culture conditions. We mainly found that ammonium was consumed faster than nitrate when provided separately, and was preferentially utilized over nitrate when both N compounds were supplied at 1:2, 1:1 and 2:1 molarity ratios. Besides, N-deficiency caused decreases in growth, energy production, antioxidative capacity and investment in photosynthate transport but increased energy consumption. Growing on ammonium produced a similar cell yield as nitrate, but with a reduced investment in nutrient transport and assimilation; yet at high concentrations ammonium exhibited inhibitory effects. These findings together have important implications in N-nutrient regulation of coral symbiosis. In addition, we identified ten highly and stably expressed genes as candidate reference genes, which will be potentially useful for gene expression studies in the future.

Purification, Characterization, Optimization and Evaluation of Potential Bioactivity of Exopolysaccharides of Curvularia lunata

Exopolysaccharides (EPS) are high-molecular-weight polysaccharides secreted by microorganisms; these polysaccharides have incredible applications in pharmaceutical industries. These are homopolymers or heteropolymers with a wide diversity of structures, capable of modifying the sensory properties of foods. Exopolysaccharides (EPSs) are recognized as high-value bio-macromolecules for the last two decades. Exopolysaccharides (EPS) are carbohydrate polymers present on the surface of many bacteria. These products, including pullulan, scleraglucan, and botryosphaeran, have several applications in industries, pharmaceuticals, medicine, foods etc. Although EPSs are highly relevant, to date, information concerning their biosynthesis is scarce, and an extensive search for new fugal species that can produce novel EPSs is still needed. In most cases, the molecular weight variations and sugar compositions of EPSs are dependent on culture medium composition and different physical conditions provided during fermentation. In this study, the selection of nutrients for the EPS production from Curvularia lunata under submerged batch-culture conditions was made using the Plackett - Burman design matrix method. Subsequently, the optimal condition was determined using Response Surface Methodology (RSM). Thus, produced EPS was characterized by using thermogravimetric analysis (TGA). The identification of components at the molecular level was made using the GC-MS analysis. It showed the presence of glucose and mannitol and good thermal stability. Their functional groups were determined using the FTIR analysis. It was further studied for its anti-cancer properties, antioxidant and antimicrobial activities. Based on the studies done, it was evident that EPS was found to be with good antimicrobial, antioxidant activity, and anti-cancer activity effective against HeLa cells.

Decolorization of Synthetic Dyes by Aspergillus flavus Strain EF-3 Isolated from Textile Dyeing Sludge

Treatment of textile and dyeing wastewater using indigenous dye-degrading microorganisms is considered to be a sustainable bioremediation strategy. In the present study, a dye-decolorizing fungal strain was isolated from dyeing sludge and identified as Aspergillus flavus strain EF-3 on the basis of cultural, morphological and internal transcribed spacer (ITS) region sequencing. The fungal strain was tested to decolorize five commercially available textile reactive dyes and dye mixture as well. The decolorization efficiencies were ranged from 62% to 100% for 100 mg/L concentration of each dye in Czapek Dox broth within 6 days of incubation at 27°C, and 120 rpm batch-culture conditions. The dyes Novacron Brilliant Blue FN-R, Bezema Yellow S8-G and dye mixture were completely decolorized within the stipulated time period. The nature of decolorization was found to be co-metabolic, i.e., dyes were not used as the sole source of energy for fungal growth and required an external co-substrate, sucrose for induction of decolorization process. The dye removal mechanism involves initial biosorption of the dye in fungal mycelium followed by subsequent biodegradation to colorless end-product. The findings of the present study provide valuable insights to desgin effective biological treatment for dyeing wastewater using the fungus Aspergillus flavus.
 Bangladesh J Microbiol, Volume 37 Number 1 June 2020, pp 7-13

Diatom-Derived Polyunsaturated Aldehydes Are Unlikely to Influence the Microbiota Composition of Laboratory-Cultured Diatoms.

Diatom-derived oxylipins, including polyunsaturated aldehydes (PUA), are considered to have infochemical, allelochemical and bacteriostatic properties, with plausible roles as grazing deterrents and regulators of inter- and intraspecific competition. However, the extent and mechanisms of how PUA influence diatom–bacteria interactions remain unresolved. In this study, impacts on the diversity of the associated bacterial communities (microbiota) of two contrasting Skeletonema marinoi strains (a PUA and a non-PUA producer) were investigated under three nitrate conditions in batch culture. Further, the response of the culture microbiota was studied when spiked with PUA at ecologically relevant concentrations (86nM octadienal and 290nM heptadienal). Of the 741 identified OTUs, Proteobacteria was the most abundant phylum (62.10%), followed by Bacteroidetes (12.33%) and Firmicutes (6.11%). Escherichia/Shigella were the most abundant genera for all treatments. Similar communities were present in both spiked and non-spiked cultures suggesting they can tolerate PUA exposure at realistic concentrations. This study suggests that PUA are not major drivers of diatom–bacteria interactions in laboratory cultures.

Utilization of various forms of nitrogen and expression regulation of transporters in the harmful alga Heterosigma akashiwo (Raphidophyceae)

Nitrogen (N) is an essential nutrient for phytoplankton growth. There is ample evidence that N enrichment promotes harmful algae blooms (HABs) but molecular mechanisms regulating N-nutrient uptake and metabolism are not so clear, especially for the raphidophyte Heterosigma akashiwo, which forms ichthyotoxic HABs in many coastal waters. In this study, the utilization of three different chemical forms of N (nitrate, ammonium, and urea) by H. akashiwo CCMA 369 was investigated in batch culture conditions. Results showed that H. akashiwo grew well on all three N compounds, and the highest cell yield occurred in the NH4+ culture group. Reverse transcription quantitative PCR analysis revealed that the expression of high-affinity NO3− transporter (NIT), NH4+ transporter (AMT) and high-affinity urea active transporter (DUR3), were significantly up-regulated under N-limitation compared to the N-replete control. The mRNA levels of AMT and DUR3 also displayed a clear diel rhythm, with high levels at midnight. In addition, NH4+ addition (5 μM) did not depress the transcript abundance of any of the three N transporters. Compared with the co-occurring immobile diatom Skeletonema costatum, the high expression of AMT in dark period in H. akashiwo is consistent with its diel vertical migration behavior, which may promote N-nutrient acquisition from deeper layers and give advantages for H. akashiwo to form blooms.

Substrate Optimization in Baby Hamster Kidney Cell Culture for Foot and Mouth Disease Virus Vaccine Using the Taguchi Method.

Cell culture is one of the most commonly used techniques in the production of biological products. Many physical and chemical parameters may affect cell growth and proliferation. This study was conducted to investigate the effect of chemical components as supplements using the experimental design method, which aimed at reducing the number of experiments. For this purpose, supplements including chemical components using four levels, with three replications in suspension and batch culture conditions, were examined for 72 hours using the Taguchi experimental design method. From the experiments, it was concluded that the culture media composition had a significant impact on final cell count and pH. High concentrations of different media composition alone were insufficient to ensure higher cell count. According to the results, this insufficiency was associated with an increase of 20% in the number of final cells. In the majority of cultures, the number of final cells at 48 hours increased relative to the number of final cells at 24 hours after culturing the cells.

PSVI-8 Detoxification of zearalenone and deoxynivalenol by Biofix® Plus PRO in a molasses-based liquid feed supplement in an in vitro rumen batch culture system

Abstract Supplementation of animal feeds with mycotoxin binders is routinely used to mitigate mycotoxin challenges. Adsorption efficiency of a binder is dependent on the structure and chemical properties of the mycotoxin, binder, and the complex gastrointestinal environment. Limited adsorption of non-polar mycotoxins has driven innovation of mitigation strategies including biotransformation or detoxification of mycotoxins by microorganisms and enzymatic activity. The current objective was to measure the efficacy of Biofix® Plus PRO (BPL) within a molasses-based liquid feed supplement to detoxify zearalenone (ZEN) and deoxynivalenol (DON) in an in vitro rumen batch culture system. Twelve reactors were filled with 1 L of liquid (50% rumen fluid, 30% reverse osmosis water, 20% synthetic saliva) and 10 g of feed (43% chopped hay, 57% corn-soy-based concentrate). Three treatments were tested in quadruplicate: Control (CON; liquid mixture only), Mycotoxin (MTX; CON + mycotoxin culture material to achieve concentration of 100 ppb ZEN and 1000 ppb DON), and BPL (MTX + 20 g/L of molasses-based liquid feed supplement including 1.65% of BPL). Samples were taken at 0 (molasses-based liquid feed added to BPL reactors after 15 min of incubation), 1, 3, 6, and 24 h and analyzed for ZEN and DON by HPLC. Results were averaged by treatment. No ZEN or DON was detected at any time point in CON group. After 3 h, ZEN was reduced by 91% and DON reduced by 87% in BPL group, with no detectable mycotoxins by 6 h. After 24 h, ZEN and DON were reduced 35% and 28%, respectively in MTX group due to natural microbial activity of the rumen fluid. Overall, the addition of Biofix® Plus PRO reduced ZEN and DON levels under the rumen batch culture conditions.

Glutamine-induced filamentous cells of Pseudomonas mediterranea CFBP-5447T as producers of PHAs.

Polyhydroxyalkanoates (PHAs) are considerable biopolymers that have gained an increasing biotechnological interest in different applications, although their industrial production presents several limitations. Filamentous bacterial cells could represent a possible strategy to increase PHA yield, since more abundant PHA inclusions can be stored in elongated than in rod-shaped cells. At first, we determined the optimal batch culture conditions to induce filamentation in Pseudomonas mediterranea CFBP-5447T, using glutamine, glycerol, glucose, and sodium octanoate, as the sole carbon source, at low- (100 rpm) or high- (250 rpm) shaking speeds. Successively, a fermentative process was set up using glutamine in a co-metabolic strategy with glycerol, and the PHAs production was compared in rod-shaped and filamentous cells. High glutamine concentrations (from 28 to 56 mM) were able to induce alone filamentation, whereas at lower glutamine concentrations (5-10 mM), the shaking speeds became critical to allow or not filamentous phenotype. PHA granule production was higher in filamentous than in rod-shaped cells, when glycerol (46.6 mM) was added to glutamine (5 mM) in co-metabolism, and fermentation was performed at a low-shaking speed. After extraction and precipitation, PHA yield was about two times higher in filamentous than that rod-shaped cells. Our results provide new insights into filament-inducing conditions and indicate a potential use of filamentous P. mediterranea CFBP-5447T cells to increase PHA yield. These findings could have great advantages in PHAs recovering during downstream processes, since the harvesting of elongated cells is much less time-consuming and energy expensive than required with rod-shaped cells.

Cytoplasmic conductivity as a marker for bioprocess monitoring: Study of Chinese hamster ovary cells under nutrient deprivation and reintroduction.

The ability to monitor the status of cells during nutrient limitation is important for optimizing bioprocess growth conditions in batch and fed-batch cultures. The activity level of Na+ /K+ ATPase pumps and cytoplasm ionic concentrations are directly influenced by the nutrient level, and thus, cytoplasm conductivity can be used as a markerless indicator of cell status. In this work, we monitored the change in cytoplasm conductivity of Chinese hamster ovary (CHO) cells during nutrient deprivation and reintroduction. Employing single cell dielectrophoresis, the change in cytoplasm conductivity was measured over a 48-hr period. The conditions under which the cytoplasm conductivity would recover to a normal level after nutrient reintroduction was determined. In addition, numerical simulations of cell ion flux, for different levels of Na+ /K+ ATPase pump inhibition, were used to predict the minimum conductivity expected for nutrient-deprived CHO cells. This predicted value is close to the minimum observed experimental cytoplasm conductivity for CHO cells that maintain the ability to restore the cytoplasm conductivity to the normal viable levels when nutrients are reintroduced. The recovery of starved cells was verified by reintroducing them to nutrient for 36 hr and measuring their proliferation using trypan blue exclusion assay. We conclude that cytoplasm conductivity can be used as a marker to indicate whether cells are in a recoverable state, such that the reintroduction of nutrients results in cells returning to a normal healthy state.

Isolation and characterization of Aeromonas sp. TXBc10 capable of high-efficiency degradation of octylphenol polyethoxylate from tannery wastewater

ABSTRACT Octylphenol polyethoxylate (OP n EO) is a potential endocrine-disrupting chemical. Biodegradation of OP n EO was investigated using the bacterium Aeromonas sp. TXBc10 isolated from the influent of tannery waste treatment plant in Hunan Province, China. The bacterium was capable of utilizing OP n EO as the sole source of carbon and energy while OP n EO was degraded under aerobic batch culture conditions. Microbial degradation of OP n EO was studied in batch experiments for optimization of environmental factors. The effect of initial OP n EO concentrations on the degradation was investigated between 100 and 1000 mg·L−1 and the results showed that the biodegradation process conformed to the first-order kinetic model. The optimum pH, temperature, salinity and size of inoculum for OP n EO degradation by Aeromonas sp. TXBc10 was found to be 8.0°C, 30°C, 2.5% and 2.0%, respectively. The major metabolites of OP n EO degradation were identified as short-chain octylphenol polyethoxylate without octylphenol by high-performance liquid chromatography – mass spectrometer, which indicated that sequential cleavage of single ethoxylate units to form OP n- 1EC might be the transformation mechanism of strain TXBc10. Considering that strain TXBc10 exhibits prominent advantage for biodegradation of OP n EO, it might serve as a promising candidate for in situ remediation of OP n EO and compounds with similar structure in tannery wastewater.

DETERMINATION OF GROWTH KINETICS AND BIOCHEMICAL COMPOSITION OF Nitzschia palea (Kützing) W. Smith ISOLATED FROM FRESHWATER SOURCES IN TURKEY

This study was performed in order to bring out a detailed information on growth dynamics and biochemical determination of the diatom species Nitzschia palea (Kützing) W. Smith under batch culture conditions in order to pave the way for further studies. The study material was isolated from a fresh water sample collected from Ankara, Turkey. The diatoms were cultured in Allen medium for 168 hours and the growth dynamics were determined by cell density and dry weight analyses. Specific growth rate, duplication time of the culture and biochemical compositions were also investigated. Molecular characterization of the N. palea strain was performed by applying Fourier Transform Infrared Spectroscopy. The cell density and the dry biomass of the culture at the end of the 168 hours incubation period was determined as 2.0x106±1.0x105cells/mL and 0.212±0.041 g L-1, respectively. The algal specific growth rate was found as 0.010 h-1 at 96-h and the doubling time was calculated as 68 h-1. The protein content was measured as 41.21%, carbohydrate content as 21.74%, lipid content as 16.84% and ash content as 19.88%. These results indicated that N. palea may be used in different fields of industries, especially in biodiesel production.

High-throughput screening of antibody-expressing CHO clones using an automated shaken deep-well system.

Biopharmaceutical protein manufacturing requires the highest producing cell lines to satisfy current multiple grams per liter requirements. Screening more clones increases the probability of identifying the high producers within the pool of available transfectant candidate cell lines. For the predominant industry mammalian host cell line, Chinese hamster ovary (CHO), traditional static‐batch culture screening does not correlate with the suspension fed‐batch culture used in manufacturing, and thus has little predictive utility. Small scale fed‐batch screens in suspension culture correlate better with bioreactor processes but a limited number of clones can be screened manually. Scaled‐down systems, such as shaken deep well plates, combined with automated liquid handling, offer a way for a limited number of scientists to screen many clones. A statistical analysis determined that 384 is the optimal number of clones to screen, with a 99% probability that six clones in the 95th percentile for productivity are included in the screen. To screen 384 clones efficiently by the predictive method of suspension fed‐batch, the authors developed a shaken deep‐well plate culturing platform, with an automated liquid handling system integrating cell counting and protein titering instruments. Critical factors allowing deep‐well suspension culture to correlate with shake flask culture were agitation speed and culture volume. Using our automated system, one scientist can screen five times more clones than by manual fed‐batch shake‐flask or shaken culture tube screens and can identify cell lines for some therapeutic protein projects with production levels greater than 6 g/L. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1460–1471, 2018

Influence of bacteria on the response of microalgae to contaminant mixtures

When microalgae are exposed to contaminants, the role of associated bacteria within the phycosphere, the microenvironment surrounding algal cells, remains largely unknown. The present study investigated the importance of algae-associated bacteria on the responses of microalgae growth to metallic and organic toxicant exposure. The effects of a polluted sediment elutriate, and of metal or pesticide mixtures at environmentally relevant concentrations (<10 μg L−1) were assessed on the growth of two microalgae strains: Isochrysis galbana, a prymnesiophyte, and Thalassiosira delicatula, a centric diatom. Both cultures were maintained as axenic or bacterized under similar conditions in batch cultures. In axenic conditions, the metal mixture addition at low concentrations alleviated limitation of growth by metals for T. delicatula relative to control, but inhibited I. galbana growth at highest concentration. In similar axenic conditions, both T. delicatula and I. galbana growth were negatively inhibited by pesticide mixture at concentrations as low as 10 ng L−1. The bacterial diversities associated with the two microalgae strains were significantly different (Bray–Curtis dissimilarity greater than 0.9) but their impact on microalgae growth was similar. The presence of bacteria reduced algal growth rate by ca. 50% compared to axenic cultures, whereas no significant effect of sediment elutriate, metal or pesticide mixtures was noticed on non-axenic algal growth rates. These results show that bacteria may have a negative effect on algal growth but can reduce pesticide toxicity or metal availability to algae.

Bioflocculation Activity in Harvesting System: A Biotechnology Approach for Microalgae Biomass

A study on Ankistrodesmus sp.—a freshwater green microalgae species—as a bioflocculant based on its physicochemical properties and flocculation rate has been carried out. The molecular identification via 16S rDNA showed 99% resemblance of this green microalga to the Ankistrodesmus fumigatus strain. The optimum batch culture condition for the bioflocculant production was initiated by 10% inoculum (v/v). The low-concentrated bioflocculant of 10% (v/v) is considered as thermostable with a high flocculation rate to harvest the biomass of Chlorella sp. at a pH range of 5 to 9. The source of molasses, the mixture of yeast extract were used as the optimum sources of carbon and Ammonium sulfate were used as the optimum sources of nitrogen in the growth medium. Ankistrodesmus sp. bioflocculant has a high flocculation efficiency over a wide range of pH (5–9) with a low dose requirement of 10% v/v at 25°C. Hence, it is immensely competitive to promote the economic viability of the production process. Accordingly, Ankistrodesmus sp. bioflocculant has a high potential to be applied on an industrial scale in tropical regions as it does not require additional production cost.

Enhanced saturated fatty acids accumulation in cultures of newly-isolated strains of Schizochytrium sp. and Thraustochytriidae sp. for large-scale biodiesel production

Heterotrophic marine protists (Thraustochytrids) have received increasingly global attention as a renewable, sustainable and alternative source of biodiesel because of their high ability of saturated fatty acids (SFAs) accumulation. Yet, the influence of extrinsic factors (nutrients and environmental conditions) on thraustochytrid culture and optimal conditions for high SFAs production are poorly described. In the present study, two different thraustochytrid strains, Schizochytrium sp. PKU#Mn4 and Thraustochytriidae sp. PKU#Mn16 were studied for their growth and SFAs production profiles under various conditions (carbon, nitrogen, temperature, pH, KH2PO4, salinity, and agitation speed). Of the culture conditions, substrates (C and N) source and conc., temperature, and agitation speed significantly influenced the cell growth and SFAs production of both strains. Although both the strains were capable of growth and SFAs production in the broad range of culture conditions, their physiological responses to KH2PO4, pH, and salinity were dissimilar. Under their optimal batch culture conditions, peak SFAs productions of 3.3g/L and 2.2g/L with 62% and 49% SFAs contents (relative to total fatty acids) were achieved, respectively. The results of 5-L fed-batch fermentation under optimal conditions showed a nearly 4.5-fold increase in SFAs production (i.e., 7.5g/L) by both strains compared to unoptimized conditions. Of the two strains, the quality of biodiesel produced from the fatty acids of PKU#Mn4 met the biodiesel standard defined by ASTM6751. This study, to the knowledge of the authors, is the first comprehensive report of optimal fermentation conditions demonstrating enhanced SFAs production by strains belonging to two different thraustochytrid genera and provides the basis for large-scale biodiesel production.

Microbial single-cell analysis in picoliter-sized batch cultivation chambers

Microfluidics has enabled various research projects in the field of microbial single-cell analysis. In particular, single-use microfluidic cultivation devices combined with automated time-lapse imaging provide powerful approaches for analyzing microbial phenomena at the single-cell level. High spatiotemporal resolution facilitates individual cell identification and tracking, delivering detailed insights into areas like phenotypic population heterogeneity, which can be highly relevant to the fate of a microbial population and may negatively impact the efficiency of biotechnological fermentations. New tools need to be developed to access the origin of population heterogeneity and understand its functional role. In this study, we present a microfluidic device for batch cultivations inside picoliter-sized cultivation chambers that can be reversibly isolated from continuous medium supply. Therefore, the cultivation broth is simply replaced by a continuous flow of humidified air, removing any medium residue along the supply channels but preserving five picoliters of cultivation medium inside the cultivation chambers in a highly parallel manner. Living cells can grow inside our microfabricated batch chambers, which can accommodate up to several hundred cells. The chamber height approximately matches the diameter of a single cell, facilitating cell growth in monolayers that are ideal for image-based cell analysis. We successfully demonstrated the growth of Escherichia coli during continuous medium perfusion and batch cultivation conditions. As expected, the cells grew exponentially under continuous medium influx until the maximum chamber capacity was reached, but when they were cultivated under batch conditions, cellular growth underwent an exponential phase, followed by a stationary phase with obvious morphological changes.

Digital Image Analysis of Yeast Single Cells Growing in Two Different Oxygen Concentrations to Analyze the Population Growth and to Assist Individual-Based Modeling.

Nowadays control of the growth of Saccharomyces to obtain biomass or cellular wall components is crucial for specific industrial applications. The general aim of this contribution is to deal with experimental data obtained from yeast cells and from yeast cultures to attempt the integration of the two levels of information, individual and population, to progress in the control of yeast biotechnological processes by means of the overall analysis of this set of experimental data, and to assist in the improvement of an individual-based model, namely, INDISIM-Saccha. Populations of S. cerevisiae growing in liquid batch culture, in aerobic and microaerophilic conditions, were studied. A set of digital images was taken during the population growth, and a protocol for the treatment and analyses of the images obtained was established. The piecewise linear model of Buchanan was adjusted to the temporal evolutions of the yeast populations to determine the kinetic parameters and changes of growth phases. In parallel, for all the yeast cells analyzed, values of direct morphological parameters, such as area, perimeter, major diameter, minor diameter, and derived ones, such as circularity and elongation, were obtained. Graphical and numerical methods from descriptive statistics were applied to these data to characterize the growth phases and the budding state of the yeast cells in both experimental conditions, and inferential statistical methods were used to compare the diverse groups of data achieved. Oxidative metabolism of yeast in a medium with oxygen available and low initial sugar concentration can be taken into account in order to obtain a greater number of cells or larger cells. Morphological parameters were analyzed statistically to identify which were the most useful for the discrimination of the different states, according to budding and/or growth phase, in aerobic and microaerophilic conditions. The use of the experimental data for subsequent modeling work was then discussed and compared to simulation results generated with INDISIM-Saccha, which allowed us to advance in the development of this yeast model, and illustrated the utility of data at different levels of observation and the needs and logic behind the development of a microbial individual-based model.