Final Cell Count Research Articles

Probiotic Encapsulation: Bead Design Improves Bacterial Performance during In Vitro Digestion (Part 2: Operational Conditions of Vibrational Technology).

The development of functional foods is a viable alternative for the prevention of numerous diseases. However, the food industry faces significant challenges in producing functional foods based on probiotics due to their high sensitivity to various processing and gastrointestinal tract conditions. This study aimed to evaluate the effect of the operational conditions during the extrusion encapsulation process using vibrating technology on the viability of Lactobacillus fermentum K73, a lactic acid bacterium with hypocholesterolemia probiotic potential. An optimal experimental design approach was employed to produce sweet whey-sodium alginate (SW-SA) beads with high bacterial content and good morphological characteristics. In this study, the effects of frequency, voltage, and pumping rate were optimized for a 300 μm nozzle. The microspheres were characterized using RAMAN spectroscopy, scanning electron microscopy, and confocal laser scanning microscopy. The optimal conditions for bead production were found: 70 Hz, 250 V, and 20 mL/min with a final cell count of 8.43 Log10 (CFU/mL). The mean particle diameter was 620 ± 5.3 µm, and the experimental encapsulation yield was 94.3 ± 0.8%. The INFOGEST model was used to evaluate the survival of probiotic beads under gastrointestinal tract conditions. Upon exposure to in vitro conditions of oral, gastric, and intestinal phases, the encapsulated viability of L. fermentum was 7.6 Log10 (CFU/mL) using the optimal encapsulation parameters, which significantly improved the survival of probiotic bacteria during both the encapsulation process and under gastrointestinal conditions compared to free cells.

The role of yeast propagation aeration for subsequent primary fermentation with respect to performance and aroma development

SummaryDuring the primary fermentation of beer, the yeast simultaneously carries out a series of processes such as cell growth, pH shift, and the formation and degradation of essential flavour components. Harvested yeast from a previous fermentation can be used to inoculate the fermentation or fresh cells can be produced through aerobic propagation. This study investigated the influence of different aeration conditions during Saccharomyces pastorianus ssp. carlsbergensis propagation on the cell count development and the production of secondary metabolites during the subsequent primary beer fermentation. Propagations were conducted by applying six different dissolved oxygen concentrations, and the cells were used as inoculum for the subsequent fermentation. Cell count, pH shift, and the development of key aroma compounds were monitored throughout the primary fermentation to evaluate any difference between the conducted fermentations. The outcomes revealed significant distinctions between fermentations using yeast propagated under elevated oxygen levels and those propagated under reduced oxygen levels. Cells propagated using lower oxygen concentrations showed earlier cell growth with 40% lower final cell counts, resulting in 50% reduced biomass yields. Additionally, lower oxygen concentrations during propagation led to lower pH shifts during primary fermentation with 20% more higher alcohols and elevated formation of acetaldehyde, and esters.

Sustainable cultivation of Porphyridium cruentum via agro-industrial by-products: A study on biomass and lipid enhancement

This study explored the cultivation of Porphyridium cruentum using beet molasses and corn steep liquor (CSL) as alternative agri-waste substrates. The objective was to enhance the biomass and lipid production of P. cruentum, known for its potential as an industrial EPA producer. Using a Box-Behnken Design (BBD) as part of the response surface methodology (RSM), we investigated the effects of beet molasses, CSL, and F/2 nutrients on the microalga's cultivation. The results demonstrated a significant influence of these factors on the final cell count and lipid content over an 8-day cultivation period. Specifically, optimal growth conditions were identified at approximately 1.78 g/L of molasses and 1.89 g/L of CSL, yielding a cell count of 12.1 × 106 cells/mL and a lipid content of 24.48%. Validation experiments reaffirmed these findings, with observed results closely aligning with predicted values. Optimized media achieved also a significant higher content of carotenoids and phycobiliproteins compared to the standard media. This research highlights the potential of using agro-industrial by-products for large-scale cultivation of P. cruentum, offering a sustainable approach for enhancing lipid and pigment production.

Assessment of Encapsulated Probiotic Lactococcus lactis A12 Viability Using an In Vitro Digestion Model for Tilapia.

Probiotics face harsh conditions during their transit through the gastrointestinal tract (GIT) of fish because of low-pH environments and intestine fluid. Therefore, the evaluation of probiotic viability under simulated gastrointestinal conditions is an important step to consider for probiotic supplementation in fish feed prior to in vivo trials. Therefore, this study aimed to evaluate the effect of stomach and intestinal simulated conditions on the viability of encapsulated Lactococcus lactis A12 using an in vitro digestion model for tilapia. A Box Behnken design was used to evaluate the potential effect of three factors, namely stomach pH, residence time in the stomach, and enzyme quantity, on the viability of encapsulated Lactococcus lactis A12. As the main results, low pH (4.00), long residence time (4 h), and enzyme quantity (2.68 U of total protease activity) led to lower final cell counts after the phases of the stomach and intestine. Encapsulated probiotic bacteria showed higher viability (p < 0.05) and antibacterial activity (p < 0.05) against the pathogen Streptococcus agalactiae than non-encapsulated bacteria. The results suggest that L. lactis A12 survives in GIT conditions and that the proposed in vitro model could be used to explore the viability of probiotic bacteria intended for fish feed supplementation.

Development of a functional dulce de leche (milk jam) with prebiotic carbohydrates and Lacticaseibacillus rhamnosus GG

A functional dulce de leche (DL) was developed by incorporating prebiotic carbohydrates (inulin and maltodextrin) and probiotic Lacticaseibacillus rhamnosus ATCC 53103 (LGG) (either as free cells, or encapsulated in pectin beads with or without prebiotics). Encapsulation improved the viability of LGG cells in DL during refrigerated storage, and its survival under simulated gastrointestinal conditions. Final cell counts in all DL samples after 6 months were >106 CFU g−1. Incorporation of carbohydrates in DL and probiotic encapsulation state had no significant effect on colour (L∗a∗b∗), rheological and mechanical properties. Although encapsulation state caused a significant effect on acceptability of DL, all samples scored above the minimum acceptability level, with more than 80% of consumers acceptance. Storage time had a significant effect (only after 2–3 months) on DL properties (except b∗ and adhesiveness), causing an increase in rheological properties, a decrease in texture parameters, and a decrease in L∗ and a∗.

To gate or not to gate: Revisiting drinking water microbial assessment through flow cytometry fingerprinting

Flow cytometry has been utilized for over a decade as a rapid and reproducible approach to assessing microbial quality of drinking water. However, the need for specialized expertise in gating—a fundamental strategy for distinguishing cell populations—introduces the potential for human error and obstructs the standardization of methods. This work conducts a comprehensive analysis of various gating approaches applied to flow cytometric scatter plots, using a dataset spanning a year. A sensitivity analysis is carried out to examine the impact of different gating strategies on final cell count results. The findings show that dynamic gating, which requires user intervention, is essential for the analysis of highly variable raw waters and distributed water. In contrast, static gating proved suitable for more stable water sources, interstage sample locations, and water presenting a particularly low cell count. Our conclusions suggest that cell count analysis should be supplemented with fluorescence fingerprinting to gain a more complete understanding of the variability in microbial populations within drinking water supplies. Establishing dynamic baselines for each water type in FCM monitoring studies is essential for choosing the correct gating strategy. FCM fingerprinting offers a dynamic approach to quantify treatment processes, enabling options for much better monitoring and control. This study offers new insights into the vagaries of various flow cytometry gating strategies, thereby substantially contributing to best practices in the water industry. The findings foster more efficient and reliable water analysis, improving of standardizing methods in microbial water quality assessment using FCM.

B-142 Development and Clinical Validation of Artificial Intelligence Assisted Flow Cytometry Diagnosis for Acuteleukemia

Abstract Background Flow cytometry is one of the most commonly used diagnostic tools in hematopathology. Manual flow cytometry data analysis is vulnerable to substantial inter-operator variation and typically takes a technologist 20 min to analyze a case. In the last decade, artificial intelligence (AI) technology has advanced dramatically and become widely used in image processing. Previously, we created an AI-assisted workflow for classifying white blood cell subsets in peripheral blood. The goal of this study is to evaluate the automated flow cytometry analysis system for diagnosing acute leukemia. Methods A total of 117 cases were retrieved from the archived diagnostic cases, consisting of three diagnostic categories: 20 normal cases, 37 acute lymphoblastic leukemia and 60 acute myeloid leukemia. Flow cytometry data manually analyzed by a pathologist is the gold standard. The same flow cytometry data was processed by the flow cytometry analysis software DeepFlow® using proprietary AI algorithm. The final diagnosis and blast cell count of AI and manual analysis are compared by Pearson correlation coefficient. Results The AI-assisted blast cell enumeration showed moderate correlation as compared to manual evaluation (r = 0.8, Fig. 1). Manual adjustment for enumeration of CD34 negative blast population with uncommon immunophenotype is required for 7 cases, including 4 CD34−/CD117+ acute myeloid leukemia and 3 CD34−/TDT+ lymphoblastic leukemia. The AI analysis effectively recognized cell populations and provided correct diagnoses on 94% of cases (110/117). With pathologists’ review, all cases were classified into the correct diagnostic categories. Conclusion Our study demonstrated that AI can accurately recognize the flow cytometry pattern of leukemia and make the proper diagnosis. Additional training for CD34 negative blast gating and other uncommon blast immunophenotypes is being conducted. Ultimately, AI will decrease the demand on manual analysis by technologists and pathologists, standardize the analysis process, and provide accurate diagnosis.

토마토 농가 환경에서 분리한 Bacillus cereus의 열저항성 분석

Bacillus cereus (B. cereus) is a spore forming bacteria with a highly adhesive ability on the food surface and which expresses concern about commercial sterilization due to heat resistance. In this study, the heat challenge was performed at 60℃ for 60 min to derive the importance of the inactivation of bacteria by time during heating 14 isolates of B. cereus from the tomato farm environment. As a result, the final cell counts of B. cereus reduced after heating were –3.6 Log CFU/mL on average (minimum: –2.8 Log CFU/mL, maximum: –4.7 Log CFU/mL). Reduced cell counts of B. cereus after heating for 60 min ranged from 0.5 Log CFU/mL to 2.4 Log CFU/mL. Remained B. cereus after heating can recover in an appropriate environment, and cause foodborne illness. B. cereus existed in the farm environment during the cultivation stage of agricultural products can be transferred to agricultural products through various routes such as soil, toilet handles and worker gloves, thus, the management of B. cereus in a farm environment is essential. In addition, improper sterilization, manufacturing, processing and packaging in the food processing stage cannot be expected to inhibit the complete growth of foodborne pathogenic bacteria, and the risk of foodborne illness can be increased. Therefore, thorough management of these processes is considered important.

Long-term outcomes of small-incision cataract surgery in patients with uveitis.

To evaluate the long-term outcomes of manual small-incision cataract surgery (MSICS) in eyes with uveitis. Patients who underwent MSICS for uveitic cataract from 2009 to 2019 were retrospectively evaluated. Visually significant cataract and presence of less than five cells per high-power field in the anterior chamber for a minimum of 3 months were the prerequisites for surgery. Patients with follow-up less than 9 months were excluded. After exclusion, 283 eyes of 264 patients were evaluated. The mean age of patients was 44.3 ± 11.3 years. The mean follow-up duration was 22 ± 11.5 months. The mean surgical time was 11.2 ± 3.2 min. One hundred and seventy-two eyes (60.8%) had anterior uveitis, 78 (27.5%) had posterior uveitis, and 33 (11.7%) had panuveitis. At the final follow-up, 253 eyes (88.4%) had corrected distance visual acuity (CDVA) better than 0.6 log of minimum angle of resolution (LogMAR) unit. The final endothelial cell counts were significantly (analysis of variance [ANOVA], P = 0.001) lower in eyes with human leukocyte antigen (HLA)-B27-associated uveitis and in eyes with idiopathic anterior uveitis. Patients on systemic corticosteroids had significantly better (P = 0.031) final visual acuity than those without preoperative corticosteroids. Recurrent uveitis (43.8%), Posterior capsule opacification (PCO) (19.4%), glaucoma (8.5%), cystoid macular edema (CME; 13.5%), and Epiretinal membrane (ERM) (5.6%) were the frequent complications. A significantly worse (ANOVA, P = 0.001) visual prognosis was seen in patients with Vogt-Koyanagi-Harada disease (VKH), sarcoidosis, acute posterior multifocal placoid pigment epitheliopathy (APMPPE), and serpiginous choroiditis. MSICS is safe in most cataracts due to uveitis and results in improvement in CDVA at 9 months. Posterior capsule opacification, macular edema, persistent uveitis, etiology of uveitis, and use of preoperative steroids significantly influenced the visual outcome.

Banana leathers as influenced by polysaccharide matrix and probiotic bacteria

Concerns abouth diet-health relationships have led many people to include healthier snacks in their diets, including those with functional (including probiotic) properties. This study was focused on development of probiotic-loaded banana leathers. Two probiotic bacteria (the spore-forming Bacillus coagulans and the conventional non-spore-forming Lactobacillus acidophilus) and two polymeric matrices (digestible cassava starch and non-digestible bacterial cellulose - BC) have been used. The presence of probiotic bacteria (mainly L. acidophilus) reduced the tensile strength, elastic modulus and shear force of the leathers, while the BC-based leathers were stronger, stiffer and more resistant to shear stress than the starch-based ones. While a high probiotic viability was kept on fruit leathers loaded with B. coagulans during drying and room-temperature storage, those loaded with L. acidophilus suffered high viability losses upon drying, which was ascribed to osmotic stress. The nature of the biopolymeric matrix has not significantly influence the bacterial viability losses along processing and storage, or the final viable cell count released into the intestine (as assessed using an INFOGEST static in vitro simulated digestion model). The banana leathers loaded with B. coagulans were well accepted, irrespectively of being produced from BC or starch, although some negative comments on the texture and flavor of the BC-based ones have been more frequent than with the starch-based ones.

Potential of Acidithiobacillus ferrooxidans to Grow on and Bioleach Metals from Mars and Lunar Regolith Simulants under Simulated Microgravity Conditions.

The biomining microbes which extract metals from ores that have been applied in mining processes worldwide hold potential for harnessing space resources. Their cell growth and ability to extract metals from extraterrestrial minerals under microgravity environments, however, remains largely unknown. The present study used the model biomining bacterium Acidithiobacillus ferrooxidans to extract metals from lunar and Martian regolith simulants cultivated in a rotating clinostat with matched controls grown under the influence of terrestrial gravity. Analyses included assessments of final cell count, size, morphology, and soluble metal concentrations. Under Earth gravity, with the addition of Fe3+ and H2/CO2, A. ferrooxidans grew in the presence of regolith simulants to a final cell density comparable to controls without regoliths. The simulated microgravity appeared to enable cells to grow to a higher cell density in the presence of lunar regolith simulants. Clinostat cultures of A. ferrooxidans solubilised higher amounts of Si, Mn and Mg from lunar and Martian regolith simulants than abiotic controls. Electron microscopy observations revealed that microgravity stimulated the biosynthesis of intracellular nanoparticles (most likely magnetite) in anaerobically grown A. ferrooxidans cells. These results suggested that A. ferrooxidans has the potential for metal bioleaching and the production of useful nanoparticles in space.

Optimizing Commercial Manufacturing of Tisagenlecleucel for Patients in the US: A 4-Year Experiential Journey

Background:Tisagenlecleucel (Kymriah) is an autologous CD19-directed CAR-T-cell therapy, approved in Aug-2017 for treating children and young adults with relapsed/refractory (r/r) acute lymphoblastic leukemia and in May-2018 for treating adults with r/r diffuse large B-cell lymphoma. Post-approval, a key goal has been to upscale and continuously improve manufacturing success and turnaround time in the commercial settings to meet the needs of a global patient population. Here we report accrued experience from our 4-year journey of optimizing the commercial tisagenlecleucel manufacturing process at the US site (Morris Plains, NJ), for faster and successful delivery to patients in the US.Methods:As reported previously, the tisagenlecleucel manufacturing process includes leukapheresis of the patient's peripheral blood mononuclear cells, enrichment and activation of T cells, transduction of the lentiviral vector containing the anti-CD19 CAR transgene, activation with anti-CD3/CD28 antibody-coated beads, expansion in cell culture, washing, and formulation of the viable cells into a cryoformulation medium. The final product is then cryopreserved, shipped back to the treatment center and infused to patients (Tyagarajan, 2020). Use of cryopreserved leukapheresis material as the starting point in commercial manufacturing is unique to tisagenlecleucel; this allows flexibility in terms of scheduling leukapheresis when a patient's health is optimal to provide T cells, and also helps offset logistical challenges (Tyagarajan, 2019).Results:As of Jun-2021, tisagenlecleucel has been manufactured for >5000 patients worldwide, enabled by Novartis's significantly increased global manufacturing footprint at six sites strategically located across six countries (US, France, Switzerland, Germany, Japan and Australia) and a global treatment network of >340 certified centers, including 127 centers in the US.Specifically for the US manufacturing site, between Dec-2020 and Jun-2021, 376 patients in the US had starting material available for manufacturing. Overall, the manufactured product was available for shipment for 98% of patients (shipping success rate [SSR]). The commercial manufacturing success rate (MSR) was 96%, with an out-of-specification (OOS) rate of <3% and no OOS for viability. All ten OOS batches were released for infusion as benefit:risk assessment was positive. Manufacturing was cancelled for two patients upon physician's request.Immediate manufacturing capability without waiting time was available on receipt of all apheresis starting materials. The median time from start of manufacturing to shipping was 20 days. As is evident, the COVID-19 pandemic did not appear to have significantly affected the success rate or manufacturing turnaround time.These latest success metrics, reflecting significant improvements from 2018 to 2021 in MSR (69% to 96%), SSR (93% to 98%), and overall OOS rate (26% to 2%) including viability OOS rate (from 25% to 0%), are a result of upscaling the manufacturing capabilities, enhancements with hospitals focusing on optimizing apheresis collection and cryopreservation procedures, and continuous evaluation and improvement of the manufacturing process since tisagenlecleucel was first launched (Figure). Two key process and analytical improvements that were considered to have improved robustness of manufacturing and testing processes, reduced OOS rates, and minimized variability in turnaround time were introduced towards the end of 2020. Firstly, a simplified sample preparation procedure for final product cell count and viability measurement, which is more reflective of final product at infusion. Secondly, an alternate serum source (5% plasma-derived human AB serum [PD hABs]) which further improves process robustness with a trend towards improved growth and higher peak cell counts.Conclusions:Tisagenlecleucel's current global commercial manufacturing footprint and treatment network are well-positioned to meet anticipated future increase in demand for CAR-T therapies. Recent process improvements have significantly increased the MSR (to 96%) and SSR (to 98%), and immediate product availability for patients in need of CAR-T cells. Ongoing and upcoming process improvements are anticipated to further reduce the throughput time, thus allowing more patients faster access to CAR-T therapy. [Display omitted] DisclosuresRodrigues: Novartis: Current Employment. Duran: Novartis: Current Employment. Eschgfaeller: Novartis: Current Employment. Kuzan: Novartis: Current Employment. Habucky: Novartis: Current Employment.

Potential of Prebiotic D-Tagatose for Prevention of Oral Disease.

Recent studies have shown phenotypic and metabolic heterogeneity in related species including Streptococcus oralis, a typical oral commensal bacterium, Streptococcus mutans, a cariogenic bacterium, and Streptococcus gordonii, which functions as an accessory pathogen in periodontopathic biofilm. In this study, metabolites characteristically contained in the saliva of individuals with good oral hygiene were determined, after which the effects of an identified prebiotic candidate, D-tagatose, on phenotype, gene expression, and metabolic profiles of those three key bacterial species were investigated. Examinations of the saliva metabolome of 18 systemically healthy volunteers identified salivary D-tagatose as associated with lower dental biofilm abundance in the oral cavity (Spearman’s correlation coefficient; r = -0.603, p = 0.008), then the effects of D-tagatose on oral streptococci were analyzed in vitro. In chemically defined medium (CDM) containing D-tagatose as the sole carbohydrate source, S. mutans and S. gordonii each showed negligible biofilm formation, whereas significant biofilms were formed in cultures of S. oralis. Furthermore, even in the presence of glucose, S. mutans and S. gordonii showed growth suppression and decreases in the final viable cell count in a D-tagatose concentration-dependent manner. In contrast, no inhibitory effects of D-tagatose on the growth of S. oralis were observed. To investigate species-specific inhibition by D-tagatose, the metabolomic profiles of D-tagatose-treated S. mutans, S. gordonii, and S. oralis cells were examined. The intracellular amounts of pyruvate-derived amino acids in S. mutans and S. gordonii, but not in S. oralis, such as branched-chain amino acids and alanine, tended to decrease in the presence of D-tagatose. This phenomenon indicates that D-tagatose inhibits growth of those bacteria by affecting glycolysis and its downstream metabolism. In conclusion, the present study provides evidence that D-tagatose is abundant in saliva of individuals with good oral health. Additionally, experimental results demonstrated that D-tagatose selectively inhibits growth of the oral pathogens S. mutans and S. gordonii. In contrast, the oral commensal S. oralis seemed to be negligibly affected, thus highlighting the potential of administration of D-tagatose as an oral prebiotic for its ability to manipulate the metabolism of those targeted oral streptococci.

Applying fluorescence in situ hybridization to aquatic systems with cyanobacteria blooms: Autofluorescence suppression and high‐throughput image analysis

AbstractCyanobacterial Harmful Algal Blooms (CyanoHABs) are expanding geographically in both fresh and marine water bodies due to coastal eutrophication and global climate change and are restructuring the microbial ecology of these systems. Cyanobacterial autofluorescence can pose a significant impediment to accurately identifying prokaryotic taxonomic groups in environmental samples using fluorescence in situ hybridization (FISH). This can hinder our ability to accurately quantify, and therefore fully understand ecological changes. As abundances of FISH target cells and autofluorescent cells can often be of the same the order of magnitude, simply subtracting average autofluorescent cell concentrations—determined from enumerating unhybridized samples—yields apparent concentrations of target cells with unacceptably large analytical uncertainty. Here we present a CuSO4/EtOH chemical pretreatment protocol that significantly reduces undesirable autofluorescence in hybridized environmental samples. We apply a novel data filtration routine to FISH images that efficiently removes residual autofluorescent cells from final cell counts. We then subject images to an automated image analysis routine that accurately enumerates probe‐positive cells. This method is inexpensive and easy to implement as part of a routine FISH workflow. By applying this method to cyanobacteria rich samples, we can better understand how microbial community changes are contributing to globally changing biogeochemical cycles.

PSIII-7 Evaluating sow colostrum somatic cell count as an indicator for colostrum composition or litter characteristics

Abstract Sow colostrum is essential during early piglet life to provide passive immunity via immunoglobulins and leukocytes.The somatic cell population in colostrum and milk consists of leukocytes and epithelial cells. Somatic cell count (SCC) of milk is commonly used as an indicator of cow milk quality and health status, but not commonly measured in lactating gilts or sows. An experiment was conducted to evaluate the relationship between colostrum SCC and colostrum composition or litter performance. A total of 194 frozen (-20℃) colostrum samples from Large White × Landrace females were evaluated for SCC. Cells were pelleted and washed twice by centrifugation (10 min. at 400 x g; 4℃) in Phosphate Buffered Saline (PBS; pH 7.2, LifeTechnologies). The resulting cell sample was labelled with a nuclear dye (LDS751) and cells counted via micro-capillary bench-top flow cytometer (Guava EasyCyte Plus, Millipore). Final SCC (cells/mL) was calculated by dividing sample cell count (cells/sample) by original sample volume (mL/sample). Data was analyzed for relationship between SCC and response variables using Pearson correlation. A pairwise comparison was used to evaluate SCC by parity category (gilts vs. sows). Results were considered significant at P&amp;lt; 0.05. Final somatic cell counts ranged from 5.8×104 to 2.9×106 cells/mL. Sows had decreased (P=0.033) SCC compared to gilts (3.0 ×105 vs. 3.7 ×105). There was no evidence for a relationship (P &amp;gt;0.05) between SCC and piglet weight at 24h or weaning, 24h litter gain, 24h or pre-wean mortality, sow backfat or body weight. Colostrum total solids, protein, lactose, and immunoglobulin G concentration showed no evidence for relationship (P &amp;gt;0.05) to SCC. Colostrum fat showed a weak positive correlation (P=0.018, R=0.18) with SCC. In conclusion, colostrum SCC is lower in sows than gilts, but does not appear to correlate to colostrum composition or litter performance.

No Effect of Microgravity and Simulated Mars Gravity on Final Bacterial Cell Concentrations on the International Space Station: Applications to Space Bioproduction.

Microorganisms perform countless tasks on Earth and they are expected to be essential for human space exploration. Despite the interest in the responses of bacteria to space conditions, the findings on the effects of microgravity have been contradictory, while the effects of Martian gravity are nearly unknown. We performed the ESA BioRock experiment on the International Space Station to study microbe-mineral interactions in microgravity, simulated Mars gravity and simulated Earth gravity, as well as in ground gravity controls, with three bacterial species: Sphingomonas desiccabilis, Bacillus subtilis, and Cupriavidus metallidurans. To our knowledge, this was the first experiment to study simulated Martian gravity on bacteria using a space platform. Here, we tested the hypothesis that different gravity regimens can influence the final cell concentrations achieved after a multi-week period in space. Despite the different sedimentation rates predicted, we found no significant differences in final cell counts and optical densities between the three gravity regimens on the ISS. This suggests that possible gravity-related effects on bacterial growth were overcome by the end of the experiment. The results indicate that microbial-supported bioproduction and life support systems can be effectively performed in space (e.g., Mars), as on Earth.

Isolation, identification, and selection of strains as candidate probiotics and starters for fermentation of Swedish legumes.

BackgroundThe non-dairy sector is growing, fermented alternatives to dairy are sparse. Adapted starter cultures to substituting raw materials needs to be developed.ObjectiveAims of this study were to isolate, identify, and phenotypically characterize lactic acid bacteria (LAB) that inhabit Swedish legumes, and assess properties necessary for selecting strains with the ability to ferment a bean beverage and with potential health beneficial properties.DesignIsolates of presumed LAB were obtained from legumes collected at Öland, Sweden. Strain diversity was assessed by repetitive polymerase chain reaction (rep-PCR). The strains were identified using matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry (MALDI-TOF MS). Species belonging to Enterococcus were predominant along with Pediococcus and closely related Bacillus. Strains were tested for tolerance to low pH, phenol, and bile as well as their bile salt hydrolase (BSH) activity. In addition, Enterococcus strains were tested for antibiotic resistance, and Pediococcus strains for their ability to ferment a bean beverage.ResultsFrom the 25 strains characterized, five were found resistant to low pH, bile, and phenol, suggesting that they can survive a passage through the gastrointestinal tract (GIT) and hence potentially exert beneficial effects in the host. These are suggested for further investigation on specific host-beneficial properties. Two of these, belonging to Pediococcus pentosaceus, were able to ferment a bean beverage without any added nutrients, indicating that the Pediococcus strains are well adapted to the bean substrate. One of the P. pentosaceus strains were also able to markedly improve the reduction of phytate by the phytase-producing yeast strain Pichia kudriavzevii TY1322 during co-fermentation as well as increase the final cell count of the yeast strain.ConclusionStrain isolation and characterization performed in this study aids in selecting starter cultures for legume fermentation. Nutritional properties can be improved by co-fermentation with yeast indicating that novel nutritious fermented non-dairy products could be developed.

Comparison of the Photoautotrophic Growth Regimens of Chlorella sorokiniana in a Photobioreactor for Enhanced Biomass Productivity

Microalgae have a wide industrial potential because of their high metabolic diversity and plasticity. Selection of optimal cultivation methods is important to optimize multi-purpose microalgal biotechnologies. In this research, Chlorella sorokiniana AM-02 that was isolated from a freshwater lake was cultured under various high photosynthetic photon flux density (PPFD) conditions and CO2 gas levels in standard Bold’s basal medium (BBM). Furthermore, a wide range of nitrate levels (180–1440 mg L−1) was tested on the growth of C. sorokiniana. Microalgae growth, pigment concentration, medium pH, exit gas composition, as well as nitrate, phosphate, and sulfate levels were measured during an experimental period. The preferred high PPFD and optimal CO2 levels were found to be 1000–1400 μmol photons m−2 s−1 and 0.5–2.0% (v/v), respectively. The addition of nitrate ions (up to 1440 mg L−1) to the standard growth medium increased final optical density (OD750), cell count, pigment concentration, and total biomass yield but decreased the initial growth rate at high nitrate levels. Our findings can serve as the basis for a robust photoautotrophic cultivation system to maximize the productivity of large-scale microalgal cultures.

Potential novel nutritional beverage using submerged fermentation with Bacillus subtilis WX-17 on brewers’ spent grains

Food processing generates side streams that are not fully utilized and typically treated as waste materials. One of such food by-product, brewers’ spent grains (BSG) are disposed in huge quantities from the beer industry annually. Submerged fermentation of BSG using Bacillus subtilis WX-17, without supplementary components, is herein employed. The fermentation products were extracted in the liquid phase, resulting in a potential novel nutritional beverage containing Bacillus subtilis WX-17. Bacillus subtilis WX-17, was still viable after a period of 6 weeks with a final cell count of 9.86 log CFU/mL. Gas chromatography-mass spectrophotometry (GC-MS) was employed for identification of the metabolites produced from the growth of Bacillus subtilis WX-17. Seven essential amino acids and citric acid cycle (TCA) intermediates were found to have increased significantly (p < 0.05) whereas all carbohydrates decreased significantly (p < 0.05) in the beverage after submerged fermentation. Additionally, antioxidant activity quantified using DPPH radical scavenging activity, increased by 2.08-fold while total phenolic content increased from 125.7 ± 0.74 μg/mL to 446.74 ± 1.26 μg/mL. The results proved the potential of employing submerged fermentation of BSG using Bacillus subtilis WX-17 to produce a novel and highly nutritious beverage.

Applying uncertainty analysis to assess the variation of operator performance when manually gating Flow Cytometry data

Background & Aim The use of Flow Cytometry (FC) for cell identification and counting is prevalent within the biomanufacture of Advanced Therapy Medicinal Products (ATMPs) both in clinical centres and biomanufacturing facilities. Operators involved in the use of FC and the analysis of the resultant datasets have the option of manually gating cell clusters or using a range of automated software platforms (supplied by FC instrument manufacturers, or third party). It is well known that many operators prefer to manually gate on the basis of speed and simplicity, however with increasing data complexity (especially when dealing with multi-colour FC instruments) there is significant potential for variation in final cell count to increase as a function of operator subjectivity. Understanding this relationship between increasing data dimensionality and operator subjectivity during manual gating techniques is important because it allows process facilities to comprehend and quantify the potential for process error as a function of incorrect measurement data. This research has specifically considered how uncertainty analysis can be used to better define operator variation over and beyond traditional measures such as the Coefficient of Variation (CV), where CV is typically only representative of the final gating process and does not provide resolution of understanding at each gating step. Methods, Results & Conclusion Three stages of cell model complexity were designed and trialled (3, 5 and 8 step), measuring variation in manual gating sequence steps to obtain a target cell population, with operators selected from academic and industrial research, and national measurement laboratory process team environments. Increasing operator variation was found as a function of increasing FC data complexity. When considering 3 step, 5 step and 8 step FC analysis, operator CV was found to range from 6% to 57% (3 step to 8 step data respectively). At the same time uncertainty analysis was applied to each gating analysis step providing clear definition of variation at each step allowing the generation of inter- and intra-operator variation statistics. Resultant combined uncertainty statements expressed at a one sigma coverage factor (67%) showed variation from 12% (3 step data) through to 34% (8 step data). Process improvement for manually gated FC analysis can therefore take advantage of this more detailed variation analysis framework and provides a benchmark for future comparison with FC automated software platforms.