Continuous Culture Conditions Research Articles

P-219 Does a dry or humid incubation environment affect continuous and undisturbed culture media protocols?

Abstract Study question Can continuous embryo culture medium maintain a safe and effective osmolality in dry and humidified incubators under both light viscosity and heavy viscosity oil overlay? Summary answer Undisturbed embryo culture is possible in 25uL and 100uL media volumes with both light and heavy oil and in both dry and humidified environments. What is known already Ideal osmolality for pre-implantation embryos is 255-295 mOsm/kg. Much debate surrounds continuous embryo culture, without refreshing of the medium, providing a suitable concentration of osmoles during embryo culture in both humidified and dry incubators. Meanwhile, ideal pH or measure of free hydrogen ions available in the embryo culture medium is 7.20-7.50. Contemporary knowledge that undisturbed embryos are able to regulate their own needs during culture has replaced belief that embryos require specific and varying environmental pH induced at different developmental stages. Thus, environmental pH needs to be maintained and stable in order to optimize health and outcomes of resulting blastocysts. Study design, size, duration Osmolality, pH, and mass were measured in humidified and dry incubators over 8 days of continuous embryo culture conditions. Four dishes each of 25uL and 100uL drops of embryo culture medium (CSCM-NXC) were analyzed per incubator type (Non-humidified – Miri, Humidified – Astec). Additionally, each micro-volume was analyzed under both Light Oil for Embryo Culture and Heavy Oil for Embryo Culture (Irvine Scientific). In total, 2352 data points for osmolality, pH, and mass were examined. Participants/materials, setting, methods A private IVF laboratory conducted the testing with no patient samples introduced to the embryo culture media. To mitigate early evaporative loss, dish preparation was strictly time and temperature controlled. Osmolality was measured in triplicate on days 0-7 using Advanced Micro-Osmometer Model 330 (Advanced Instruments). Mass measures were taken in triplicate on days 0-7 using the Torbal AGZN120 Analytical Balance. pH was measured in triplicate using the i-STAT 300 (Abbott) on days 0,3,5,7. Main results and the role of chance The pH in 25uL and 100uL drops under light oil and heavy oil remained well within suitable ranges and stable when compared day to day, from day 0 (M = 7.276, σ = 0.011) through day 7 (M = 7.303, σ = 0.011). The average aggregate pH overall was 7.290 (σ = 0.012). Aggregate osmolality, measured as milliosmoles per kilogram (mOSM/kg), over the course of incubation in all conditions also remained well within acceptable safety range (M = 277mOsm/kg, σ = 5). Dry incubator incubation exhibits a 7% increase from day 0 to day 6 (M = 270mOsm/kg, σ = 4 and M = 289mOsm/kg, σ = 5) compared to a 2% increase in humidified incubation (M = 269mOsm/kg, σ = 5 and M = 274mOsm/kg, σ = 5) regardless of oil type or microdrop size, both remaining within accepted limits. Additionally, when monitoring mass in grams (g) in 25uL as a function of evaporative loss, humidified incubation reduces from 12.6346g to 12.5566g (day 0 to day 7). In dry incubation, there is negligible change day 0 to day 7 (12.6543g and 12.6538g). Similarly, in 100uL, the change in humidified incubators is 13.37222g to 13.25025g (day 0 to day 7) and dry incubation reduces from 13.3236g to 13.2963g. The changes in mass of prepared culture dishes maintains consistency with the study findings for osmolality. Limitations, reasons for caution As CSCM-NXC is for use through day 6 of embryo culture, the extended simulated embryo culture length is for data collection purposes only. There is no developmental data to include in this study. Additional studies must be conducted in order to explore any differences when embryos are present in culture. Wider implications of the findings The ability to culture embryos continuously in dry incubator environments with micro-volumes of culture medium is widely debated. These many data point study has shown that safe and efficacious embryo culture can be undertaken undisturbed, therefore relaxing some of the burdensome and labor-intensive processes in laboratories employing this equipment. Trial registration number not applicable

Streamlining the biodesulfurization process: development of an integrated continuous system prototype using Gordonia alkanivorans strain 1B.

Biodesulfurization is a biotechnological process that uses microorganisms as biocatalysts to actively remove sulfur from fuels. It has the potential to be cleaner and more efficient than the current industrial process, however several bottlenecks have prevented its implementation. Additionally, most works propose models based on direct cultivation on fuel, or batch production of biocatalysts followed by a processing step before application to batch biodesulfurization, which are difficult to replicate at a larger scale. Thus, there is a need for a model that can be adapted to a refining process, where fuel is being continuously produced to meet consumer needs. The main goal of this work was to develop the first bench-scale continuous biodesulfurization system that integrates biocatalyst production, biodesulfurization and fuel separation, into a single continuous process, taking advantage of the method for the continuous production of the biodesulfurization biocatalysts previously established. This system eliminates the need to process the biocatalysts and facilitates fuel separation, while mitigating some of the process bottlenecks. First, using the bacterium Gordonia alkanivorans strain 1B, continuous culture conditions were optimized to double biocatalyst production, and the produced biocatalysts were applied in batch biphasic biodesulfurization assays for a better understanding of the influence of different factors. Then, the novel integrated system was developed and evaluated using a model fuel (n-heptane + dibenzothiophene) in continuous biodesulfurization assays. With this system strain 1B surpassed its highest biodesulfurization rate, reaching 21 μmol h-1 g-1. Furthermore, by testing a recalcitrant model fuel, composed of n-heptane with dibenzothiophene and three alkylated derivatives (with 109 ppm of sulfur), 72% biodesulfurization was achieved by repeatedly passing the same fuel through the system, maintaining a constant response throughout sequential biodesulfurization cycles. Lastly, the system was also tested with real fuels (used tire/plastic pyrolysis oil; sweet and sour crude oils), revealing increased desulfurization activity. These results highlight the potential of the continuous biodesulfurization system to accelerate the transition from bench to commercial scale, contributing to the development of biodesulfurization biorefineries, centered on the valorization of sulfur-rich residues/biomasses for energy production.

Effects of Cashew Nut Shell Extract on Ruminal Fermentation and Nutrient Digestibility under Continuous Culture

The overall objective of this study was to determine the dose response to four levels of cashew nut shell extract in a granulated form (CNSE, containing 59% anacardic acid and 18% cardol) on culture pH, rumen fermentation metabolites, and apparent nutrient digestibility in continuous culture fermenters. The study was conducted as a generalized randomized complete block design with four treatments and four replications per treatment. The four treatments were randomly assigned to eight fermenters for two incubation runs of 10 d. Treatments consisted of (1) Control (CO, no CNSE), (2) Control plus 100 ppm of CNSE, (3) Control plus 200 ppm of CNSE, and (4) Control plus 300 ppm of CNSE. Fermenters were fed 52 g/d (DM basis) of a total mixed ration (TMR; 17.0% crude protein (CP), 29.7% NDF, and 29.9% starch), divided between two feedings at 0800 and 2000 h. The apparent digestibility of dry matter (DM), organic matter (OM), and neutral detergent fiber (NDF) were not affected by CNSE supplementation. Similarly, CNSE had no effect on culture pH, total volatile fatty acids (VFA) or individual VFA molar proportions. These results suggest that at the dosages evaluated in this study, CNSE has no impact on the rumen fermentation profile and the apparent nutrient digestibility under continuous culture conditions.

Long-Term Characteristics of Human-Derived Biliary Organoids under a Single Continuous Culture Condition

Organoids have been used to investigate the three-dimensional (3D) organization and function of their respective organs. These self-organizing 3D structures offer a distinct advantage over traditional two-dimensional (2D) culture techniques by creating a more physiologically relevant milieu to study complex biological systems. The goal of this study was to determine the feasibility of establishing organoids from various pediatric liver diseases and characterize the long-term evolution of cholangiocyte organoids (chol-orgs) under a single continuous culture condition. We established chol-orgs from 10 different liver conditions and characterized their multicellular organization into complex epithelial structures through budding, merging, and lumen formation. Immunofluorescent staining, electron microscopy, and single-nucleus RNA (snRNA-seq) sequencing confirmed the cholangiocytic nature of the chol-orgs. There were significant cell population differences in the transcript profiles of two-dimensional and organoid cultures based on snRNA-seq. Our study provides an approach for the generation and long-term maintenance of chol-orgs from various pediatric liver diseases under a single continuous culture condition.

Milk Exosomes Select Mutations That Decrease the Toxicity of Clostridioides difficile

ObjectivesBackground: Exosomes are natural nanoparticles that play an essential role in cell-to-cell communication. Exosomes are present in all body fluids and their concentration is particularly high in milk. We previously found that bovine milk exosomes (BMEs) select polymorphisms and mutations in gut bacteria, potentially affecting the synthesis of microbial metabolites. Clostridioides difficile (C. difficile) is the primary cause of infectious diarrhea in the United States and it is unknown whether BMEs impact its pathogenicity through the selection of mutations. Objective: Assess the effect of chronic BME exposure on C. difficile growth and pathogenicity. MethodsC. difficile was cultured in BME-supplemented and -free media under continuous culture conditions for 21 days. Strains were isolated from both conditions. Doubling times and maximal density were assessed by monitoring optical density (OD600). Toxicity was assessed by Vero cell rounding and qRT-PCR of toxin genes (tcdA and tcdB) mRNA. Whole-genome sequencing was used to identify mutations in C. difficile strains. Effects of gene mutations on growth and toxicity were determined by overexpression of the mutants in C. difficile. Mann-Whitney test was used for statistical analysis; P < 0.05 was considered significant. ResultsBMEs selected for mutations that reduced doubling time by 15% (P < 0.05), decreased tcdA and tcdB production by 92% and 93%, respectively, (P < 0.05) and relative toxin activity reduced by 74% (p = 0.055). BME-selected strains contained a 25-bp deletion in the gene encoding the glucose-specific PTS transporter subunit IIBCA (ptsG). As a result, the translated ptsG (ptsG-indel) protein is truncated by 49 amino acids with 5 mismatches compared with wild-type. Overexpression of ptsG-indel in a wild-type strain of C. difficile increased maximum OD600 by 34% (P < 0.05), lowered relative toxin activity by 88% (P < 0.001), and reduced tcdB expression by 94% (P < 0.05) compared to C. difficile that overexpress wild-type ptsG. ConclusionsBMEs decreased the pathogenicity of C. difficile. Decreased pathogenicity was likely due to mutations in ptsG selected by growth in the presence of BMEs. Funding SourcesNIH P20GM104320, NIFA 2020–67,017-30,834, USDA Hatch and W-4002. J.Z is a consultant for PureTech Health, Inc.

Arsenosugars and arsenolipids are formed simultaneously by the unicellular alga

Environmental context Arsenic is a globally distributed element, occurring in various chemical forms with toxicities ranging from harmless to highly toxic. We conducted 48-h cell culture experiments under batch and continuous conditions using the ubiquitous marine unicellular alga Dunaliella tertiolecta and evaluated the alga’s arsenic metabolome over time. We found that the alga first methylates the inorganic As taken up from the surrounding water, and then further metabolises the intermediate simultaneously into more complex organo-arsenic molecules like sugars and lipids. These time series experiments are valuable pieces in the puzzle of how algae bio-metabolise arsenic, and in our understanding of the global arsenic cycle. Rationale The uptake of arsenate by algae from oceanic waters and its transformation to arsenosugars and arsenolipids is well established, but the biosynthetic pathways remain largely unknown. Methodology We investigated these pathways by using time-series experiments over 48 h to follow the formation of organoarsenic species from arsenate-enriched medium (15 µg As L−1) by the unicellular alga Dunaliella tertiolecta cultured under batch and continuous culture conditions. We used complementary mass spectrometry methods for the determination and quantification of 14 arsenic species; an additional three species could be quantified but remained unidentified. Results The alga rapidly methylated the arsenate to dimethylarsinate (DMA), which then served as the precursor to arsenosugars and arsenolipids; the concentrations of these complex organoarsenicals increased throughout the experiments accompanied by a concomitant reduction in DMA concentrations. The pattern of compounds formed by the alga was similar for both batch and continuous cultures, but the concentrations were 2–3-fold higher in the continuous culture samples and the increases with time were much clearer. Discussion The data suggest that the arsenosugars and the arsenolipids were mostly formed simultaneously from DMA, although there was an indication that the arsenic phospholipids were at least partly also being formed from the arsenosugars. Overall, the data are consistent with a direct biosynthesis of DMA from arsenate by D. tertioleta, and thereafter a non-specific incorporation of DMA into commonly available alga metabolites encompassing various sugars and lipids.

An experimental and in silico analysis of Lacticaseibacillus paracasei isolated from whey shows an association between lactate production and amino acid catabolism.

The production of lactic acid from agroindustry waste products, such as whey, heavily relies on microorganisms within the genusLactobacillus. In this work, a genome-scale metabolic model was implemented from Vinay-Lara (iLca334_548), improved adding some enzymatic reactions and used to analyse metabolic fluxes ofLacticaseibacillus paracasei, which is aLactobacillusstrain isolated from whey used in the large-scale production of lactic acid. Overall, the highest rate of lactic acid productivity was 2.9 g l-1h-1, which equates to a dilution rate of 0.125 h-1, when continuous culture conditions were established. Restrictions on lactic acid production caused by exchange reactions, complex culture medium and intracellular metabolite concentrations were considered and included in the model. In total, theiLca334_548 model consisted of 1046 reactions and 959 metabolites, and flow balance analysis better predicted lactate flux than biomass. The distribution of fluxes exhibited an increase in lactate formation as biomass decreased. This finding is supported by the reactions carried out by glyceraldehyde 3-phosphate dehydrogenase, pyruvate formate lyase and ribose-5-phosphate isomerase, corroborating the modelled phenotype with experimental data. In conclusion, there is potential for the improvement of lactate production in a complex media by amino acid catabolism, especially when lactate is derived from pyruvate.

Bioethanol production of cheese whey in continuous culture

The cheese industry produces a large amount of waste, the equivalent of 9L of cheese whey for every 10L of milk (Murari et al., 2018; Pendon et al., 2021; Sar et al., 2021). Whey is composed by lactose and proteins, mainly (Murari et al., 2018; Murari et al., 2019; Tesfaw et al., 2021). In most cases, cheese whey had not added value or specific use, being discarded and causing very important environmental implications (Maruri et al., 2018; Tesfaw et al., 2021; Sar et al., 2021). In the present study, the production of bioethanol from cheese whey were analyzed, using Kluyveromyces marxianus as inoculum, under continuous culture conditions (28°C, agitation at 100 rpm, aeration at 1 vvm, feed at 8.63 mL/min), where it was established that is possible to obtain yields of 0.62 g of ethanol per g of lactose in the first 18 h, this makes it feasible for the continuous production of ethanol with cheese whey with minimal pretreatment.

Enabling high-throughput biology with flexible open-source automation.

Our understanding of complex living systems is limited by our capacity to perform experiments in high throughput. While robotic systems have automated many traditional hand‐pipetting protocols, software limitations have precluded more advanced maneuvers required to manipulate, maintain, and monitor hundreds of experiments in parallel. Here, we present Pyhamilton, an open‐source Python platform that can execute complex pipetting patterns required for custom high‐throughput experiments such as the simulation of metapopulation dynamics. With an integrated plate reader, we maintain nearly 500 remotely monitored bacterial cultures in log‐phase growth for days without user intervention by taking regular density measurements to adjust the robotic method in real‐time. Using these capabilities, we systematically optimize bioreactor protein production by monitoring the fluorescent protein expression and growth rates of a hundred different continuous culture conditions in triplicate to comprehensively sample the carbon, nitrogen, and phosphorus fitness landscape. Our results demonstrate that flexible software can empower existing hardware to enable new types and scales of experiments, empowering areas from biomanufacturing to fundamental biology.

Slow Adaptive Response of Budding Yeast Cells to Stable Conditions of Continuous Culture Can Occur without Genome Modifications.

Continuous cultures assure the invariability of environmental conditions and the metabolic state of cultured microorganisms, whereas batch-cultured cells undergo constant changes in nutrients availability. For that reason, continuous culture is sometimes employed in the whole transcriptome, whole proteome, or whole metabolome studies. However, the typical method for establishing uniform growth of a cell population, i.e., by limited chemostat, results in the enrichment of the cell population gene pool with mutations adaptive for starvation conditions. These adaptive changes can skew the results of large-scale studies. It is commonly assumed that these adaptations reflect changes in the genome, and this assumption has been confirmed experimentally in rare cases. Here we show that in a population of budding yeast cells grown for over 200 generations in continuous culture in non-limiting minimal medium and therefore not subject to selection pressure, remodeling of transcriptome occurs, but not as a result of the accumulation of adaptive mutations. The observed changes indicate a shift in the metabolic balance towards catabolism, a decrease in ribosome biogenesis, a decrease in general stress alertness, reorganization of the cell wall, and transactions occurring at the cell periphery. These adaptive changes signify the acquisition of a new lifestyle in a stable nonstressful environment. The absence of underlying adaptive mutations suggests these changes may be regulated by another mechanism.

Continuous biohydrogen production from poplar biomass hydrolysate by a defined bacterial mixture immobilized on lignocellulosic materials under non-sterile conditions

Lignocellulosic biowastes are rich in sugars and may be used as low-cost feed to produce environmentally friendly fuels such as hydrogen (H2). Poplar biomass is available in large quantities as a sugar-rich biowaste. Few studies have evaluated bacterial H2 production using well-defined mixed cultures (DMC) under non-sterile continuous culture conditions. DMC composed of high H2-producing bacteria may circumvent the potential risk of a single bacterial culture being out-competed under non-sterile conditions. The novelty of this study lies in the continuous production of H2 from poplar biomass using DMC in immobilized state under non-sterile conditions. Under optimized continuous culture conditions with DMC immobilized on coconut coir, a H2 yield of 2.83 mol/mol of hexose was steadily obtained over a period of 40 days, which was four-fold higher compared to that obtained with Bacillus thuringiensis. This approach may enable to meet the increasing demand for cleaner energy-rich fuels on a large-scale in a steady manner.

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.

Evaluation of internal reference genes in Auxenochlorella protothecoides under continuous heterotrophic culture conditions at normal, low and high temperatures

Real-time reverse quantitative transcription PCR (RT-qPCR) is a commonly used method for gene expression analysis. However, the accuracy of RT-qPCR analysis greatly depends on the stability of the reference genes selected for data normalization. Unfortunately, there have been no systematic evaluation studies on reference genes in the unicellular green alga Auxenochlorella protothecoides. Ten genes (ACT, COX6B, CYB5R4, EF1α, GAPDH, rbcL, RPL19, RPL32, RPS10 and TUA) were chosen as candidate reference genes. The expression stability of each reference gene was determined by applying geNorm, NormFinder, BestKeeper and comprehensive ranking based on geomean ranking values. In order to validate the suitability and reliability of the candidate reference genes selected for data normalization, two key functional genes related to lipid metabolism were also selected as target genes. The results revealed that RPL32 was the most stably expressed and suitable reference genes under all experimental conditions of the analyses; CYB5R4 was also a suitable reference gene under continuous heterotrophic culture condition, and RPL19 was top-ranked and stably expressed under both low and high temperature stress. These results provided a list of suitable reference genes for accurate normalization of heterotrophic A. protothecoides time-course samples under different temperature stress in qPCR experiments. This study will be of very great reference value to assess the stability of candidate reference genes in other heterotrophically grown algae.

Physiologic expansion of human heart-derived cells enhances therapeutic repair of injured myocardium

BackgroundSerum-free xenogen-free defined media and continuous controlled physiological cell culture conditions have been developed for stem cell therapeutics, but the effect of these conditions on the relative potency of the cell product is unknown. As such, we conducted a head-to-head comparison of cell culture conditions on human heart explant-derived cells using established in vitro measures of cell potency and in vivo functional repair.MethodsHeart explant-derived cells cultured from human atrial or ventricular biopsies within a serum-free xenogen-free media and a continuous physiological culture environment were compared to cells cultured under traditional (high serum) cell culture conditions in a standard clean room facility.ResultsTransitioning from traditional high serum cell culture conditions to serum-free xenogen-free conditions had no effect on cell culture yields but provided a smaller, more homogenous, cell product with only minor antigenic changes. Culture within continuous physiologic conditions markedly boosted cell proliferation while increasing the expression of stem cell-related antigens and ability of cells to stimulate angiogenesis. Intramyocardial injection of physiologic cultured cells into immunodeficient mice 1 week after coronary ligation translated into improved cardiac function and reduced scar burden which was attributable to increased production of pro-healing cytokines, extracellular vesicles, and microRNAs.ConclusionsContinuous physiological cell culture increased cell growth, paracrine output, and treatment outcomes to provide the greatest functional benefit after experimental myocardial infarction.

Equine adipose mesenchymal stem cells (eq-ASCs) appear to have higher potential for migration and musculoskeletal differentiation

Equine adipose-derived mesenchymal stem cells (eq–ASCs) possess excellent regeneration potential especially for treatment of musculoskeletal disorders. Besides their common characteristics, MSCs harvested from different species reveal some species-specific and donor-dependent behaviors. Hence, the molecular analysis of MSCs may shed more light on their future clinical application of these cells. This study aimed to investigate some behavioral aspects of eq-ASCs in vitro which may influence the efficacy of stem cell therapy. For this purpose, MSCs of a donor horse were isolated, characterized and expanded under normal culture conditions. During continuous culture condition, eq-ASCs were started to formed aggregated structures that was accompanied with the up-regulation of migratory related genes including transforming growth factor beta 1 (TGFB1) and its receptor 3 (TGFBR3), and snail family transcriptional repressor 1 (SNAI1), E-cadherin (CDH1) and β-catenin (CTNNB1). Moreover, the expression of a musculoskeletal progenitor marker, scleraxis bHLH transcription factor (SCX), was also increased after 3 days. In order to clarify the impact of TGFB signaling pathway on cultured cells, gain– and loss-of-function treatment by TGFB3 and SB431542 (TGFB inhibitor) were performed, respectively. We found that TGFB3 treatment exaggerated the aggregate formation effects, in some extend via induction of cytoskeletal actin rearrangement, while inhibition of TGFB signaling pathway by SB431542 reversed this phenomenon. Overall, our findings support the fact that eq-ASCs have an inherent capacity for migration, which was enhanced by TGFB3 treatment and, this ability may play crucial role in cell motility and wound healing of transplanted cells.

Transient exposure to oxygen or nitrate reveals ecophysiology of fermentative and sulfate-reducing benthic microbial populations.

SummaryFor the anaerobic remineralization of organic matter in marine sediments, sulfate reduction coupled to fermentation plays a key role. Here, we enriched sulfate‐reducing/fermentative communities from intertidal sediments under defined conditions in continuous culture. We transiently exposed the cultures to oxygen or nitrate twice daily and investigated the community response. Chemical measurements, provisional genomes and transcriptomic profiles revealed trophic networks of microbial populations. Sulfate reducers coexisted with facultative nitrate reducers or aerobes enabling the community to adjust to nitrate or oxygen pulses. Exposure to oxygen and nitrate impacted the community structure, but did not suppress fermentation or sulfate reduction as community functions, highlighting their stability under dynamic conditions. The most abundant sulfate reducer in all cultures, related to Desulfotignum balticum, appeared to have coupled both acetate‐ and hydrogen oxidation to sulfate reduction. We describe a novel representative of the widespread uncultured candidate phylum Fermentibacteria (formerly candidate division Hyd24‐12). For this strictly anaerobic, obligate fermentative bacterium, we propose the name ‘USabulitectum silens’ and identify it as a partner of sulfate reducers in marine sediments. Overall, we provide insights into the function of fermentative, as well as sulfate‐reducing microbial communities and their adaptation to a dynamic environment.

Bioreactor culture duration of engineered constructs influences bone formation by mesenchymal stem cells.

Perfusion culture of mesenchymal stem cells (MSCs) seeded in biomaterial scaffolds provides nutrients for cell survival, enhances extracellular matrix deposition, and increases osteogenic cell differentiation. However, there is no consensus on the appropriate perfusion duration of cellular constructs invitro to boost their bone forming capacity invivo. We investigated this phenomenon by culturing human MSCs in macroporous composite scaffolds in a direct perfusion bioreactor and compared their response to scaffolds in continuous dynamic culture conditions on an XYZ shaker. Cell seeding in continuous perfusion bioreactors resulted in more uniform MSC distribution than static seeding. We observed similar calcium deposition in all composite scaffolds over 21 days of bioreactor culture, regardless of pore size. Compared to scaffolds in dynamic culture, perfused scaffolds exhibited increased DNA content and expression of osteogenic markers up to 14 days in culture that plateaued thereafter. We then evaluated the effect of perfusion culture duration on bone formation when MSC-seeded scaffolds were implanted in a murine ectopic site. Human MSCs persisted in all scaffolds at 2 weeks invivo, and we observed increased neovascularization in constructs cultured under perfusion for 7 days relative to those cultured for 1 day within each gender. At 8 weeks post-implantation, we observed greater bone volume fraction, bone mineral density, tissue ingrowth, collagen density, and osteoblastic markers in bioreactor constructs cultured for 14 days compared to those cultured for 1 or 7 days, and acellular constructs. Taken together, these data demonstrate that culturing MSCs under perfusion culture for at least 14 days invitro improves the quantity and quality of bone formation invivo. This study highlights the need for optimizing invitro bioreactor culture duration of engineered constructs to achieve the desired level of bone formation.

Plasmodium falciparum in vitro continuous culture conditions: A comparison of parasite susceptibility and tolerance to anti-malarial drugs throughout the asexual intra-erythrocytic life cycle

The continuous culture of Plasmodium falciparum is often seen as a means to an end, that end being to probe the biology of the parasite in question, and ultimately for many in the malaria drug discovery arena, to identify means of killing the parasite in order to treat malaria. In vitro continuous culture of Plasmodium falciparum is a fundamental requirement when undertaking malaria research where the primary objectives utilise viable parasites of a desired lifecycle stage. This investigation, and resulting data, compared the impact culturing Plasmodium falciparum long term (4 months) in different environmental conditions had on experimental outcomes and thus conclusions. The example presented here focused specifically on the effect culture conditions had on the in vitro tolerance of Plasmodium falciparum to standard anti-malarial drugs, including artemisinin and lumefantrine. Historical data from an independent experiment for 3D7-ALB (5% O2) was also compared with that obtained from this study. We concluded that parasites cultured for several months in media supplemented with a serum substitute such as Albumax II® or within hyperoxic conditions (21% O2), demonstrate highly variable responses to artemisinin and lumefantrine but not all anti-malarial drugs, when compared to those cultured in human serum in combination with Albumax II® under normoxic conditions (5% O2) for the parasite.

Municipal wastewater treatment via co-immobilized microalgal-bacterial symbiosis: Microorganism growth and nutrients removal

A symbiotic microalgal-bacterial system may be an optional technology for wastewater treatment. In this study, co-immobilized of a bacterium isolated from a municipal wastewater treatment plant (Pseudomonas putida) and a microalgae Chlorella vulgaris was used in the study of cell growth and nutrient removal during wastewater treatment under batch and continuous culture conditions. Under batch culture conditions, co-immobilization treatment significantly increased the cell density of C. vulgaris and P. putida compared with other treatments. The co-immobilized treatment also showed higher removal of ammonium, phosphate and COD than any single treatment, indicating that the nutrient uptake capability of C. vulgaris and P. Putida was mutually enhanced mutually. When tested in continuous mode, the treatment with a hydraulic retention time of 24h at the organic load rate of 1159.2mgCODL−1d−1 was most appropriate for wastewater treatment.

Accumulation of biomass and four triterpenoids in two-stage cultured Poria cocos mycelia and diuretic activity in rats

Poria cocos (Schw.) Wolf, an important medicinal and food fungus, is well known in East Asia. Due to growing market demand, long cultivation period, and consumption of pine trunk during cultivation, developing alternative methods for producing P. cocos and/or its active components is of interest. In the present study, the effects of different culture methods on biomass and accumulation of four triterpenoids were investigated. The ethanol extract of fermented mycelium (EFM) was orally administered to rats. Urine output and concentrations of electrolytes (Na+, K+, and Cl−) were measured. Our results showed that mycelia grew better under continuous shaking culture condition (7.5 g DW·L−1), and higher triterpenoid levels were accumulated in two-stage culture (112 mg·L−1, 2.03%). The optimal starting time of static culture for triterpenoid yield was 4th d after shaking culture. Single administration of middle and high dose of EFM significantly increased urine output, Na+ and Cl− excretion, and Na+/K+ ratio. These results suggested that ethanol extract of cultured mycelia showed significant diuretic activity in rats and two-stage culture of P. cocos could be an alternative way to produce mycelia and triterpenoids.