Viscoelastic properties of suspended cells measured with shear flow deformation cytometry

Numerous cell functions are accompanied by phenotypic changes in viscoelastic properties, and measuring them can help elucidate higher-level cellular functions in health and disease. We present a high-throughput, simple and low-cost microfluidic method for quantitatively measuring the elastic (storage) and viscous (loss) modulus of individual cells. Cells are suspended in a high-viscosity fluid and are pumped with high pressure through a 5.8 cm long and 200 μm wide microfluidic channel. The fluid shear stress induces large, near ellipsoidal cell deformations. In addition, the flow profile in the channel causes the cells to rotate in a tank-treading manner. From the cell deformation and tank treading frequency, we extract the frequency-dependent viscoelastic cell properties based on a theoretical framework developed by R. Roscoe that describes the deformation of a viscoelastic sphere in a viscous fluid under steady laminar flow. We confirm the accuracy of the method using atomic force microscopy-calibrated polyacrylamide beads and cells. Our measurements demonstrate that suspended cells exhibit power-law, soft glassy rheological behavior that is cell cycle-dependent and mediated by the physical interplay between the actin filament and intermediate filament networks.

Study on the effect of headspace on biohydrogen production using palm oil mill effluent (POME) via immobilized and suspended growth

The world has been using fossil fuels to generate energy for centuries and has had adverse effects on the environment; hence renewable energy needs to be discovered and developed. Biohydrogen production is renewable energy since it emits no greenhouse gases and may provide clean energy. Therefore, this study aimed to investigate the optimum headspace ratio and biohydrogen production for suspended and immobilized cells using Palm Oil Mill Effluent (POME) as the fermentation substrate, while its anaerobic sludge acted as the inoculum. Five different ratios were investigated, which are 0.2, 0.3, 0.4, 0.5, and 0.6. These are equivalent to working volume (WV) of 80 mL, 70 mL, 60 mL, 50 mL, and 40 mL, respectively. The solution contained 10 % of inoculum and 90 % (v/v) of the feedstock. For immobilized cells, additional of glass beads as carrier material was added into the solution, using the ratio of 1:1 for anaerobic sludge (mL) to support carrier (g). The kinetic study was investigated using a modified Gompertz equation whereby for suspended cells, the best ratio was 0.3, with the highest biohydrogen concentration of 357.6 ppm. Meanwhile, the optimum ratio for the immobilized cell was 0.2, with the highest biohydrogen concentration of 479.3 ppm. Based on the kinetic studies, the kinetic parameters for suspended cells were: Hm = 89.8 mL, Rm = 6.8 mL/h, and λ = 0.1 hr. Meanwhile for immobilized cell, the kinetic parameters were: Hm = 73.6 mL, Rm = 6.9 mL/h and X λ 0 hr. In conclusion, selecting the suitable headspace ratio could affect the biohydrogen quality and improve the effectiveness of the production rate.

Profiling Myxococcus xanthus swarming phenotypes through mutation and environmental variation

Myxococcus xanthus is a bacterium that lives on surfaces as a predatory biofilm called a swarm. As a growing swarm feeds on prey and expands, it displays dynamic multicellular patterns such as traveling waves called ripples and branching protrusions called flares. The rate at which a swarm expands across a surface, and the emergence of the coexisting patterns, are all controlled through coordinated cell movement. M. xanthus cells move using two motility systems known as Adventurous (A) and Social (S). Both are involved in swarm expansion and pattern formation. In this study, we describe a set of M. xanthus swarming genotype-to-phenotype associations that include both genetic and environmental perturbations. We identified new features of the swarming phenotype; recorded and measured swarm expansion using time-lapse microscopy; and compared the impact of mutations on different surfaces. These observations and analyses have increased our ability to discriminate between swarming phenotypes and provided context that allow us to identify some phenotypes as improbable ‘outliers’ within the M. xanthus swarming phenome. IMPORTANCE Myxococcus xanthus grows on surfaces as a predatory biofilm called a swarm. In nature, a feeding swarm expands by moving over and consuming prey bacteria. In the laboratory, a swarm is created by spotting cell suspension onto nutrient agar in lieu of prey. The suspended cells quickly settle on the surface as the liquid is absorbed into the agar, and the new swarm then expands radially. An assay that measures the expansion rate of a swarm of mutant cells is the first, and sometimes only, measurement used to decide whether a particular mutation impacts swarm motility. We have broadened the scope of this assay by increasing the accuracy of measurements and introducing prey, resulting in new identifiable and quantifiable features that can be used to improve genotype-to-phenotype associations.

Immobilized Microalgae using Alginate for Wastewater Treatment

Organic and inorganic substances are released into the environment because of domestic, agricultural, and industrial activities which contribute to the pollution of water bodies. Removal of these substances from wastewater using conventional treatment involves high energy cost for mechanical aeration to provide oxygen for aerobic digestion system. During this process, the aerobic bacteria rapidly consume the organic matter and convert it into single cell proteins, water, and carbon dioxide. Alternatively, this biological treatment step can be accomplished by growing microalgae in the wastewater. Chlorella vulgaris immobilized in calcium alginate was used to study the removal efficiency of main nutrients in wastewater such as ammonium and phosphate that act as an important factor in microalgae growth. The immobilized cells demonstrated higher percentage of ammonium and phosphate removal of 83% and 79% respectively, compared to free-suspended cells (76% and 56%). COD removal recorded was 89% and 83% for immobilized cells and free-suspended cells, respectively. The kinetics parameters of nutrients removal for immobilized C. vulgaris in synthetic wastewater were also determined. The specific ammonium removal rates (RA) and phosphate removal rates (RP) for Chlorella vulgaris in synthetic wastewater were 8.3 mg.L-1day-1 and 7.9 mg.L-1day-1, respectively. On the other hand, the kinetic coefficient for each nutrient removal determined were kA = 0.0462 L.mg-1 day-1 NH4 and kP = 0.0352 L.mg-1 day-1 PO43-. This study proves the application of immobilized microalgae cells is advantageous to the wastewater treatment efficiency. Furthermore, optimization on the immobilization process can be conducted to further improve the nutrients removal rates which potentially can be applied in the large-scale wastewater treatment process.

Photoinactivation of Pseudomonas aeruginosa Biofilm by Dicationic Diaryl-Porphyrin

In recent years, antimicrobial photodynamic therapy (aPDT) has received increasing attention as a promising tool aimed at both treating microbial infections and sanitizing environments. Since biofilm formation on biological and inert surfaces makes difficult the eradication of bacterial communities, further studies are needed to investigate such tricky issue. In this work, a panel of 13 diaryl-porphyrins (neutral, mono- and di-cationic) was taken in consideration to photoinactivate Pseudomonas aeruginosa. Among cationic photosensitizers (PSs) able to efficiently bind cells, in this study two dicationic showed to be intrinsically toxic and were ruled out by further investigations. In particular, the dicationic porphyrin (P11) that was not toxic, showed a better photoinactivation rate than monocationic in suspended cells. Furthermore, it was very efficient in inhibiting the biofilms produced by the model microorganism Pseudomonas aeruginosa PAO1 and by clinical strains derived from urinary tract infection and cystic fibrosis patients. Since P. aeruginosa represents a target very difficult to inactivate, this study confirms the potential of dicationic diaryl-porphyrins as photo-activated antimicrobials in different applicative fields, from clinical to environmental ones.

Probiotic Lactobacillus sp. Strains Inhibit Growth, Adhesion, Biofilm Formation, and Gene Expression of Bacterial Vaginosis-Inducing Gardnerella vaginalis

Gardnerella vaginalis contributes significantly to bacterial vaginosis, which causes an ecological imbalance in vaginal microbiota and presents with the depletion of Lactobacillus sp. Lactobacillus supplementation was reported to be an approach to treat bacterial vaginosis. We investigated the applicability of three Lactobacillus sp. strains (Lactobacillus delbrueckii DM8909, Lactiplantibacillus plantarum ATCC14917, and Lactiplantibacillus plantarum ZX27) based on their probiotic abilities in vitro. The three candidate Lactobacillus sp. strains for bacterial vaginosis therapy showed distinct properties in auto-aggregation ability, hydrophobicity, adhesion to cervical epithelial cells, and survivability in 0.01% hydrogen peroxide. Lpb. plantarum ZX27 showed a higher yield in producing short-chain fatty acids and lactic acid among the three candidate strains, and all three Lactobacillus sp. strains inhibited the growth and adhesion of G. vaginalis. Furthermore, we discovered that the culture supernatant of Lactobacillus sp. exhibited anti-biofilm activity against G. vaginalis. In particular, the Lpb. plantarum ZX27 supernatant treatment decreased the expression of genes related to virulence factors, adhesion, biofilm formation, metabolism, and antimicrobial resistance in biofilm-forming cells and suspended cells. Moreover, Lactobacillus sp. decreased the upregulated expression of interleukin−8 in HeLa cells induced by G. vaginalis or hydrogen peroxide. These results demonstrate the efficacy of Lactobacillus sp. application for treating bacterial vaginosis by limiting the growth, adhesion, biofilm formation, and virulence properties of G. vaginalis.

Telomerase Activity and Myogenesis Ability as an Indicator of Cultured Turkey Satellite Cell Ability for In Vitro Meat Production

Telomerase activity is highly correlated to the proliferation capacity and immortality of cells. To evaluate the possibility of continuous culture, myoblasts were isolated from the Pectoralis thoracicus muscle of newborn turkeys and maintained in 2D (adherence based) and suspension cultures. Furthermore, adherent myoblasts were differentiated into myotubes. Telomerase activity was evaluated in all types of obtained cultures. The expression of telomerase related genes, including TERT1, TERT2, dyskerin, as well as myogenesis related genes, including myogenin, MyoD, MRF1 and MRF5 were measured. Telomerase bands were detected in both adherent and suspended cells, but they were not detected in samples from rat muscle. Myotube differentiation caused a significant reduction in the expression of TERT1, TERT2 and Dyskerin, while MyoD, Myogenin and MRF4 were upregulated in myotubes vs. myoblasts. Long-term culture of suspended myoblasts caused a significant increase in TERT1 levels, with no significant change in expression of myogenesis related genes. Overall, the results show that myoblasts are able to grow in suspension without losing their myogenic properties. Furthermore, upregulation of TERT1 indicates continued proliferation of myoblasts and generation of enough daughter cells necessary for in vitro meat production. Running title: Telomerase activity and myogenic properties of cultured Turkey satellite cells

Characterization of different biocatalyst formats for BVMO-catalyzed cyclohexanone oxidation

Cyclohexanone monooxygenase (CHMO), a member of the Baeyer-Villiger monooxygenase family, is a versatile biocatalyst that efficiently catalyzes the conversion of cyclic ketones to lactones. In this study, an Acidovorax-derived CHMO gene was expressed in Pseudomonas taiwanensis VLB120. Upon purification, the enzyme was characterized in vitro and shown to feature a broad substrate spectrum and up to 100% conversion in 6 h. Further, we determined and compared the cyclohexanone conversion kinetics for different CHMO-biocatalyst formats, i.e., isolated enzyme, suspended whole cells, and biofilms, the latter two based on recombinant CHMO-containing P. taiwanensis VLB120. Biofilms showed less favorable values for K (9.3-fold higher) and k (4.8-fold lower) compared to corresponding K and k values of isolated CHMO, but a favorable K for cyclohexanone (5.3-fold higher). The unfavorable K and k values are related to mass transfer- and possibly heterogeneity issues and deserve further investigation and engineering, in order to exploit the high potential of biofilms regarding process stability. Suspended cells showed an only 1.8-fold higher K, but 1.3- and 4.2-fold higher k and K values than isolated CHMO. This together with the efficient NADPH regeneration via glucose metabolism makes this format highly promising from a kinetics perspective.