Bacterial Cell Mass Research Articles

Binary color adsorption by zinc@magnetite nanocomposites: Robust parametric optimization using Taguchi method

AbstractThe goal of the current study was to determine the optimal level (value) of adsorption process variables for the adsorption of color from binary aqueous solution. The impact of key adsorption factors, including adsorbent dose, initial color concentration, and contact time, on the color removal effectiveness of Zinc@magnetite nanocomposites (Zin@Fe3O4(ME) produced from bacterial extracts and Zin@Fe3O4(M) produced from bacterial cell mass), has been examined. Utilizing an L9 orthogonal array (OA), average values and the SN ratio for each variable at three diverse points are calculated. ANOVA has determined the most advantageous optimum conditions for raw and SN data. According to the study, the adsorbent dose is shown to be the most important parameter for color adsorption, while contact duration is determined to be the least important factor based on Pareto chart analysis. The percentage of color removal at optimal values for Zin@Fe3O4(M) and Zin@Fe3O4(ME) nanocomposites, respectively, was found to be 81% and 73%. The optimal parameters were found to be 12.5 mg of adsorbent, 125 mg/L initial dye concentration, and 90 min of contact time at pH 7, room temperature. The confirmatory experiments conducted under optimized conditions revealed a higher percentage of color eliminated than was optimized in each test run of the Taguchi design, proving the credibility of the process variables optimized within the designated experiments.

Biogenic engineered zinc oxide nanoparticle for sulfur black dye removal from contaminated wastewater: comparative optimization, simulation modeling, and isotherms

ABSTRACT This research work aimed to isolate and culture the bacterium Bacillus paramycoides for biogenic fabrication of zinc oxide nanoparticles, specifically ZnO and ZnO-ME nanoparticles (nanoparticles fabricated from bacterial extracts only – ZnO, and from bacterial cell mass including extract – ZnO-ME). SEM investigation revealed the spherical-shaped NPs with 22.33 and 39 nm in size for ZnO and ZnO-ME, respectively. The Brunauer, Emmett, and Teller (BET) studies revealed mesoporous structure with pore diameters of 13.839 and 13.88 nm and surface area of 7.617 and 33.635 m2/gm for ZnO and ZnO-ME, respectively. Various parameters for the adsorption of sulfur black dye onto both ZnO and ZnO-ME were screened and optimized using Plackett–Burman Design (PBD), Full Factorial Design (FFD) and Central Composite Design (CCD). The results of the optimization modeling study revealed that FFD yielded the most predictable and best-fitting results among all the models studied, with R 2 values of 0.998 for ZnO and 0.993 for ZnO-ME. Notably, ZnO-ME exhibited a greater dye removal efficiency 80% than ZnO i.e., 71%, it may be due to the presence of amorphous carbon on the surface of ZnO-ME. Among the various isothermal models, the Freundlich model displayed the strongest correlation with the dye removal data, confirming the multilayer adsorption of dye on both nanoparticles and supporting physisorption. Therefore, ZnO and ZnO-ME nanoparticles have been proven as potential tools for mitigating environmental impacts associated with dye-containing wastewater.

Decolorisation of textile wastewater using biosynthesised iron oxide nanomaterials and Optimisation by Response Surface Methodology

AbstractIn this study, the bacteria (Pseudomonas sp.) was isolated and cultured for biogenic fabrication of Fe3O4 (synthesised using bacterial cell mass) and Fe3O4@ME (synthesised using bacterial extracts) nanoparticles. The biomolecules of the bacterial extract function as a reducing and capping agent, giving the nanoparticle surface negative charges, according to the FTIR and Z potential analysis. The parameters were optimised and screened for indigo blue (IB) dye adsorption onto Fe3O4 and Fe3O4@ME nanoparticles. This work used the Design of the Experimental (DoE) method rather than the conventional one‐variable‐at‐a‐time method. The central composite design (CCD) experiment consisted of 13 experimental runs, and an analysis of variance (ANOVA) was performed to analyse the results. The models for IB dye removal using Fe3O4 and Fe3O4@ME nanomaterials showed adjusted R2 values of 0.99 and 0.99 and predicted R2 values of 0.97 and 0.99, respectively, indicating good model fit. The optimum conditions required for 70 and 64 % colour removal for Fe3O4 and Fe3O4@ME nanomaterials, respectively, are observed at 0.050 gm sorbent doses at the constant room temperature and contact time of 100 min.

A First Report on the Isolation and Characterization of a Highly Potential Indigenous Mosquitocidal Bacterium (Bacillus thuringiensis Subspecies Israelensis VCRC B647) from Red Soil, India.

To control mosquito vectors causing human diseases, bacterial biopesticides are currently in use. Indeed, the recent development of resistance to these bacterial agents has impeded its applications. Under these circumstances, the search for novel bacterial agents with mosquitocidal activity is unavoidable. In this study, a novel mosquitocidal bacterium was isolated from red soils of agricultural field. The objective of this study was to isolate and identify new mosquitocidal bacteria from the natural environment. Soil samples were collected during 2021-2022 from Tirupathur district of Tamil Nadu, South India. The samples were bioprocessed for culturing the bacterial colony in a suitable culture medium (Nutrient Yeast Salt Medium), and after 72 h, the bacterial cell mass was removed and lyophilized. Bioassays (mosquito toxicity assays) were carried out to screen the bacterial colonies for mosquitocidal effect. The potential colony was further analyzed, and identified for its application in mosquito control. The new isolate screened from red soil was identified as Bacillus thuringiensis subspecies israelensis (VCRC B647) as per the ilvD gene sequence analysis. The strain was found to be potentially effective in controlling mosquito larvae, and further biochemical analyses, bacterial growth, biomass, and protein content were investigated. The new isolate did not show any toxic effect on nontarget aquatic organisms. It is significant to depict that the mosquitocidal action of this new isolate (Bti) is highly significant than the reference strain of Bti-H14. It is concluded that this is the first report that an indigenous strain of Bti VCRC B647 is very effective in mosquito control.

Evaluation of the LifeScale system for rapid phenotypic antimicrobial susceptibility testing from positive blood cultures

Abstract Objective The spread of antimicrobial resistance is a growing threat to public health, thus there is a critical need for prompt and reliable antimicrobial susceptibility testing (AST). Rapid AST may improve patient outcomes by allowing for quicker start of optimal therapy and reducing hospital length of stay, therefore decreasing overall healthcare costs. However, rapid results may not improve patient care if they are not considered equivalent to standard AST. Several platforms are under development that can provide rapid AST results; the objective of this work is to assess the performance of the new LifeScale rapid AST platform (Affinity Biosensors) that uses resonant mass measurements for calculation of antimicrobial susceptibility results. Methods Positive BACTEC blood-cultures collected from patients were included if the gram stain revealed only Gram-negative rods. Standard of care (SOC) AST results, as determined by a combination of BD Phoenix automated, Sensititre broth microdilution, and/or standard disk diffusion methods, were compared to results from direct positive blood culture testing on the LifeScale system. Minimum inhibitory concentrations (MICs) and categorical interpretations based on CLSI and/or FDA AST breakpoints were compared by microorganism and drug to calculate essential and categorical agreement for five species with pending claims on the LifeScale system: Escherichia coli (N=33), Klebsiella pneumoniae (N=10), K. oxytoca (N=5), Pseudomonas aeruginosa (N=11) and Acinetobacter sp. (N=1); tested against 13 relevant antibiotics: Amikacin, ampicillin, aztreonam, cefazolin, cefepime, ceftazidime, ceftazidime/avibactam ertapenem, gentamicin, levofloxacin, meropenem, piperacillin/tazobactam, and trimethoprim/sulfamethoxazole. Results LifeScale had 95.3% essential agreement with the SOC results, and a 93.1% categorical agreement based on CLSI M52 criteria. Of the 665 organism-drug combinations evaluated, agreement was high, with only 1 very major discrepancy, 8 major discrepancies and 37 minor discrepancies, of which 22 (47.8%) were within ±1 doubling dilution. Among these discrepancies, LifeScale MICs averaged 1 dilution higher than SOC MICs, which may reduce the risk of false-susceptible interpretations. Conclusion s: The accuracy and reliability of results from rapid AST is of utmost importance. The LifeScale technology is unique among AST platforms as it measures bacterial cell mass and counts to determine MICs for up to 14 antibiotics in ~4 h. These results suggest this platform is able to deliver rapid, actionable results to guide antimicrobial therapy for patients with bacteremia.

Fabrication, characterization and performance analysis of a two-step arsenic bio-filter column using Delftia spp. BAs29 and fired red mud pellets.

Arsenic (As) is considered to be a grave inorganic pollutant, contaminating major aquifers worldwide. In this study, a two-step approach has been designed to combat this toxic metalloid by combining a highly efficient As (III) oxidizing bacteria; Delftia sp. BAs29 and fired red mud pellets to remove the total As from groundwater including both As (III) and As (V) ions. The maximum capacity of As (III) oxidation by Delftia sp. BAs29 was seen to be 95.65% for 500ml of As contaminated groundwater using an optimized As (III) concentration of 300ppb and 6.5g of bacterial cell mass for 7days. The second step indicated the maximum As (V) adsorption capacity by the stacked red mud pellets to be 97.91% for 500ml of As contaminated groundwater using the optimized pore size of 106-125μm for 7days. The efficiency of As removal increased to 98.76% at a flow rate of 50ml/h on combining of both the steps. In addition, the morphological properties, chemical composition, and the crystal structure of the As (V) adsorbed red mud pellets were characterized. The techno-economic feasibility of this entire unit was studied using SuperPro 10 software to estimate its optimal demand and potential. Hence, it is believed that scaling up of this two-step bio-filter column can serve as a potent filtration unit to eliminate As, both at the household and industrial level in the near future.

High-throughput determination of dry mass of single bacterial cells by ultrathin membrane resonators

How bacteria are able to maintain their size remains an open question. Techniques that can measure the biomass (dry mass) of single cells with high precision and high-throughput are demanded to elucidate this question. Here, we present a technological approach that combines the transport, guiding and focusing of individual bacteria from solution to the surface of an ultrathin silicon nitride membrane resonator in vacuum. The resonance frequencies of the membrane undergo abrupt variations at the instants where single cells land on the membrane surface. The resonator design displays a quasi-symmetric rectangular shape with an extraordinary capture area of 0.14 mm2, while maintaining a high mass resolution of 0.7 fg (1 fg = 10−15 g) to precisely resolve the dry mass of single cells. The small rectangularity of the membrane provides unprecedented frequency density of vibration modes that enables to retrieve the mass of individual cells with high accuracy by specially developed inverse problem theory. We apply this approach for profiling the dry mass distribution in Staphylococcus epidermidis and Escherichia coli cells. The technique allows the determination of the dry mass of single bacterial cells with an accuracy of about 1% at an unparalleled throughput of 20 cells/min. Finally, we revisit Koch & Schaechter model developed during 60 s to assess the intrinsic sources of stochasticity that originate cell size heterogeneity in steady-state populations. The results reveal the importance of mass resolution to correctly describe these mechanisms.

A gatekeeper protein contributes to T3SS2 function via interaction with an ATPase in Vibrio parahaemolyticus

Assembly of a functional type III secretion system (T3SS) requires intricate protein-protein interactions in many bacterial species. In Vibrio parahaemolyticus, the leading cause of seafood-associated diarrheal illnesses, the gatekeeper protein VgpA is essential for T3SS2 to secrete its substrates. However, it is unknown if VgpA interacts with other core elements of T3SS2 to mediate its substrate secretion. Through bacterial two-hybrid (BACTH) analysis, we now show that VgpA physically interacts with VscN2 (an ATPase essential for T3SS function) and six other hypothetical proteins. Mutation of isoleucine to alanine at residue 175 of VgpA (VgpAI175A) abolished its ability to interact with VscN2. Importantly, complementation of a VgpA nonsense mutant (vgpA’) with VgpAI175A did not restore the ability of T3SS2 to secrete substrates, demonstrating that VgpA-VscN2 interaction is critical for the function of T3SS2. Bacterial cell fractionation and mass spectrometry analyses showed that vgpA’ resulted in significant alterations of T3SS2 protein abundance in multiple bacterial cell fractions. Particularly, VscN2 abundance in the inner membrane fraction and VscC2 abundance in the outer membrane fraction are significantly reduced in vgpA’ compared to those in WT. These results demonstrated that VgpA contributes to T3SS2 function via its interaction with VscN2 and possibly by affecting subcellular distribution of T3SS2 proteins.

To Plate or to Simply Unfreeze, That Is the Question for Optimal Plasmid Extraction.

Many molecular biology applications require fast plasmid DNA extraction, spurring multiple studies on how to speed up the process. It is regularly instructed in standard laboratory protocols to plate out frozen glycerol bacterial stocks prior to bacteria incubation in liquid media and subsequent plasmid extraction, although the rationale for this is often unexplained (other than for the isolation of single colonies). Given the commonality and importance of this laboratory operation, such a practice is time-consuming and laborious. To study the impact of this practice and the alternative direct culturing method, we investigated the association between bacterial cell mass and its potential influence on plasmid yields from the 2 methods. Our results showed no difference with preplating for 7 out of 8 plasmid constructs used in the study, suggesting that direct glycerol recovery would not lead to poorer plasmid yields. The findings support the rationale for direct glycerol recovery for plasmid extraction, without the need of an intermediate preplating step.

In vitro degradation of low-density polyethylene by new bacteria from larvae of the greater wax moth, Galleria mellonella

Three bacterial species isolated from whole body extracts of the greater wax moth larvae, Galleria mellonella, were evaluated for their ability to utilize low-density polyethylene (LDPE) as a sole carbon source invitro. These bacteria were identified as Lysinibacillus fusiformis, Bacillus aryabhattai, and Microbacterium oxydans. Their ability to biodegrade LDPE was assessed by growth curves, cell biomass production, polyethylene (PE) weight loss, and the presence of LDPE hydrolysis products in the growth media. Consortia of these bacteria with three other bacteria previously shown to degrade LDPE (Cupriavidus necator H16, Pseudomonas putida LS46, and Pseudomonas putida IRN22) were also tested. Growth curves of the bacteria utilizing LDPE as a sole carbon source revealed a peak in cell density after 24h. Cell densities declined by 48h but slowly increased again to different extents, depending on the bacteria. Incubation of LDPE with bacteria isolated from greater wax moth larvae had significant effects on bacterial cell mass production and weight loss of LDPE in PE-containing media. The bacterial consortia were better able to degrade LDPE than were the individual species alone. Gas chromatographic analyses revealed the presence of linear alkanes and other unknown putative LDPE hydrolysis products in some of bacterial culture media.

The Effect of Bifidobacterium Bifidum Supernatant and Cell Mass on the Proliferation Potential of Rat Bone Marrow-Derived Stromal Cells.

Background: Mesenchymal stem cells (MSCs) are widely used to treat various diseases, however, their proliferative potential reduces after a number of passages. It has been shown that some probiotics such as Bifidobacterium Bifidum (B. bifidum) affect the proliferation of various cell lineages. The present study aimed to investigate the effect of B. bifidum on the proliferation of rat bone marrow stromal cells (rBMSCs) and to develop a method for compensating their proliferation reduction after some passages. Methods: The present experimental study was conducted at Tehran University of Medical Sciences, Tehran, Iran, in 2017. The stromal cells were isolated from rBMSCs and their mesenchymal properties were confirmed by osteogenic and adipogenic differentiation media and staining. B. bifidum was cultured and the B. bifidum supernatant (BS) and bacterial cell mass (BCM) were extracted. The rBMSCs were treated with different concentrations of BS and BCM. The MTT assay was performed to measure the number of viable cells in the culture. Cell proliferation was analyzed using the paired-sample t test.Results: Cell proliferation increased as the concentration of bacteria was increased logarithmically (0, 0.1, 0.3, 0.9, 3, 9, 30 μL/mL). In comparison with BS, cells treated with BCM showed increased cell proliferation at lower concentrations. This effect was caused by removing the “de Man, Rogosa, and Sharpe” (MRS) broth medium from the BCM culture. The optimal concentration of bacteria with the most significant effect on rBMSCs proliferation was determined.Conclusion: A significant increase in the proliferation of stromal cells was observed; confirming the stimulatory potential of probiotics (B. bifidum) on various cells. The use of products containing probiotic bacteria can increase the proliferation potential of BMSCs.

High cell-density fermentation, expression and purification of bacteriophage lysin TSPphg, a thermostable antimicrobial protein from extremophilic Thermus bacteriophage TSP4

Recently, high cell-density (HCD) cultivation has become an important tool for production of many microbial products. However, to the best of our knowledge, no study regarding HCD fermentation, overproduction and purification of thermostable bacteriophage lysin has been reported. Here, by employing a glucose-limited fed-batch strategy, we performed high density fermentation of the host Escherichia coli BL21(DE3) cells, compared the efficiency of high pressure homogenization, ultrasonication and thermolysis in bacterial cell disruption after HCD cultivation, and purified TSPphg, a thermostable lysin derived from extremophilic bacteriophage TSP4. On the 20-L scale, the overproduction level of TSPphg was up to 67.8 ± 0.7%. In total, we obtained a broth titer of 3322.8 ± 26 mg/L TSPphg with a purity of 95.5 ± 0.7% from a bacterial cell mass of 86.3 ± 4.9 g/L after 26 h of fermentation. The overall productivity of TSPphg was 127.8 ± 1 mg/L/h. Additionally, the antimicrobial activity of purified TSPphg against both Gram-negative (Escherichia coli O157) and Gram-positive (Staphylococcus aureus) pathogenic bacteria was further confirmed by scanning electron microscope analysis. Summarily, for the first time, we have established a relatively stable and efficient HCD cultivation and purification process for recovery of thermostable lysins from extremophilic Thermus bacteriophages. Our results provide insights into the strategies for time-saving and cost-effective production of antimicrobial proteins to replace or supplement antibiotics in the current age of mounting antibiotic resistance.

Overexpression and characterization of TnCel12B, a hyperthermophilic GH12 endo-1,4-β-glucanase cloned from Thermotoga naphthophila RKU-10T

A putative cellulolytic gene (825 bp) from Thermotoga naphthophila RKU-10T was overexpressed as an active soluble endo-1,4-β-glucanase (TnCel12B), belongs to glycoside hydrolase family 12 (GH12), in a mesophilic expression host. Heterologous expression and engineered bacterial cell mass was improved through specific strategies (induction and cultivation). Hence, intracellular activity of TnCel12B was enhanced in ZYBM9 modified medium (pH 7.0) by 8.38 and 6.25 fold with lactose (200 mM) and IPTG (0.5 mM) induction, respectively; and 6.95 fold was increased in ZYP-5052 auto-inducing medium after 8 h incubation at 26 °C (200 rev min−1). Purified TnCel12B with a molecular weight of ~32 kDa, was optimally active at 90 °C and pH 6.0; and exhibited prodigious stability over a wide range of temperature (50–85 °C) and pH (5.0–9.0) for 8 h TnCel12B displayed great resistance towards different chemical modulators, though activity was improved by Mg2+, Zn2+, Pb2+ and Ca2+. Purified TnCel12B had affinity with various substrates but peak activity was observed toward barley β-glucan (1664 U mg−1) and carboxymethyl cellulose (736 U mg−1). The values of Km, Vmax, kcat, and kcatKm−1 were found to be 4.63 mg mL−1, 916 μmol mg−1min−1, 1326.7 s−1 and 286.54 mL mg−1 s−1, respectively using CMC substrate. All noteworthy features of TnCel12B make it an appropriate industrial candidate for bioethanol production and various other potential applications.

A centrifuge tube reactor for the determination of bacterial methane oxidation enrichment factors without influence of diffusion related isotope fractionation

Biotransformation of methane at landfill sites can be estimated by applying compound specific stable isotope analysis of methane from the anaerobic and the cover layer surface zone. Next to these two input parameters, merely the knowledge of the carbon isotopic fractionation of the bacterial methane oxidation in terms of the enrichment factor (ε) is required. However, many factors and conditions have been described to affect ε. These include temperature, the applied landfill cover, the type of expressed methane monooxygenase (MMO), and cell density. In this work we investigated the microbial methane oxidation with respect to temperature and type of methanotrophic enrichment culture. A newly designed setup was used to overcome potential CH4-substrate limitations such as diffusion that could affect the determined values of ε by improper and inhomogeneous mixing. The isotopic fractionation was determined based on the stable carbon isotope analysis of methane and carbon dioxide. The obtained value for isotopic fractionation was ε22°C = −0.0136 ± 0.0036. Also for the first time, bulk stable isotope analysis of bacterial cell mass was performed by flow injection analysis isotope ratio mass spectrometry.

Biosorption of Heavy Metals from Aqueous Solution by Bacteria Isolated from Contaminated Soil.

This study was carried out to analyze the heavy metals biosorption potential of bacteria isolated from soil contaminated with electroplating industrial effluents. Bacterial isolates were screened for their multi-metal biosorption potential against copper, nickel, lead, and chromium. Bacterial isolate CU4A showed the maximum uptake of copper, nickel, lead, and chromium in aqueous solution, with a biosorption efficiency of 87.16 %, 79.62%, 84.92%, and 68.12%, respectively. The bacterial strain CU4A was identified as Bacillus cereus, following 16S rRNA gene sequence analysis. The surface chemical functional groups of bacterial biomass were identified by Fourier transform infrared (FTIR) spectroscopy as hydroxyl, carboxyl, amine, and halide, which may be involved in the biosorption of heavy metals. Analysis with scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) confirmed the adsorption of metals on the bacterial cell mass. The results of this study are significant and could be further investigated for the removal of heavy metals from contaminated environments.

Microbiological Examination of Erwinia amylovora Exopolysaccharide Ooze

Fire blight, caused by the pathogen Erwinia amylovora, is the most devastating bacterial disease of pome fruit in North America and worldwide. The primary method of dispersal for E. amylovora is through ooze, a mass of exopolysaccharides and bacterial cells that is exuded as droplets from infected host tissue. During the 2013 and 2014 field seasons, 317 ooze droplets were collected from field-inoculated apple trees. Populations of E. amylovora in ooze droplets were 108 CFU/μl on average. Ooze droplets harboring larger (>108 CFU/μl) cell populations were typically smaller in total volume and had darker coloring, such as orange, red, or dark red hues. Examination of apple host tissue at the site of emergence of ooze droplets using scanning electron microscopy revealed that ooze was not exuding through natural openings; instead, it was found on erumpent mounds and small (10-μm) tears in tissue. These observations suggested that E. amylovora-induced wounds in tissue provided the exit holes for ooze extrusion from the host. Analyses of E. amylovora populations in ooze droplets and within the stems from which ooze droplets emerged indicated that approximately 9% of the total bacterial population from infected stems is diverted to ooze. Gene expression analyses indicated that E. amylovora cells in stem sections located above ooze droplets and in ooze droplets were actively expressing critical pathogenicity genes such as hrpL, dspE, and amsK. Thus, our study identified ooze as a source of large, concentrated populations of E. amylovora that emerged from the host by rupturing host tissue. Because the cells in ooze droplets are expressing genes required for pathogenesis, they are already primed for infection should they be dispersed from ooze to new infection courts.

Enhanced Production of a Recombinant Multidomain Thermostable GH9 Processive Endo-1,4-β-Glucanase (CenC) from Ruminiclostridium thermocellum in a Mesophilic Host Through Various Cultivation and Induction Strategies.

Commonly, unintentional induction and inadvertently preparing medium for engineered Escherichia coli BL21 CodonPlus (DE3)-RIPL, give poor or variable yields of heterologous proteins. Therefore, to enhance the activity and production of an industrially relevant recombinant processive endo-1,4-β-glucanase (CenC) propagated in Escherichia coli BL21 CodonPlus(DE3)-RIPL through various cultivation and induction strategies. Investigation of various growth media and induction parameters revealed that high-cell-density and optimal CenC expression were obtained in ZYBM9 medium induced either with 0.5mM IPTG/150mM lactose, after 6h induction at 37°C; and before induction, bacterial cells were given heat shock (42°C) for 1h when culture density (OD600nm) reached at 0.6. Intracellular enzyme activity was enhanced by 6.67 and 3.20-fold in ZYBM9 and 3×ZYBM9 medium, respectively, under optimal conditions. Using YNG auto-induction medium, activity was 2.5-fold increased after 10h incubation at 37°C. Approximately similar results were obtained by transferring the optimized process at the bioreactor level. Results showed that the effective process strategy is essential to enhance recombinant bacterial cell mass and enzyme production from small to large-scale. To the best of our knowledge, this is the first ever report on enhanced production of thermostable processive endo-1,4-β-glucanase cloned from Ruminiclostridium thermocellum, which is a suitable candidate for industrial applications. Graphical Abstract Flow Chart Summary of Enhanced Production of a Recombinant Multidomain Thermostable GH9 Processive Endo-1,4-β-glucanase from Ruminiclostridium thermocellum.

Antibacterial Activities of Graphene Oxide-Molybdenum Disulfide Nanocomposite Films.

Two-dimensional (2D) nanomaterials, such as graphene-based materials and transition metal dichalcogenide (TMD) nanosheets, are promising materials for biomedical applications owing to their remarkable cytocompatibility and physicochemical properties. On the basis of their potent antibacterial properties, 2D materials have potential as antibacterial films, wherein the 2D nanosheets are immobilized on the surface and the bacteria may contact with the basal planes of 2D nanosheets dominantly rather than contact with the sharp edges of nanosheets. To address these points, in this study, we prepared an effective antibacterial surface consisting of representative 2D materials, i.e., graphene oxide (GO) and molybdenum disulfide (MoS2), formed into nanosheets on a transparent substrate for real device applications. The antimicrobial properties of the GO-MoS2 nanocomposite surface toward the Gram-negative bacteria Escherichia coli were investigated, and the GO-MoS2 nanocomposite exhibited enhanced antimicrobial effects with increased glutathione oxidation capacity and partial conductivity. Furthermore, direct imaging of continuous morphological destruction in the individual bacterial cells having contacts with the GO-MoS2 nanocomposite surface was characterized by holotomographic (HT) microscopy, which could be used to detect the refractive index (RI) distribution of each voxel in bacterial cell and reconstruct the three-dimensional (3D) mapping images of bacteria. In this regard, the decreases in both the volume (67.2%) and the dry mass (78.8%) of bacterial cells that came in contact with the surface for 80 min were quantitatively measured, and releasing of intracellular components mediated by membrane and oxidative stress was observed. Our findings provided new insights into the antibacterial properties of 2D nanocomposite film with label-free tracing of bacterial cell which improve our understanding of antimicrobial activities and opened a window for the 2D nanocomposite as a practical antibacterial film in biomedical applications.

A Simple and Rapid Method for Preparing a Cell-Free Bacterial Lysate for Protein Synthesis.

Cell-free protein synthesis (CFPS) systems are important laboratory tools that are used for various synthetic biology applications. Here, we present a simple and inexpensive laboratory-scale method for preparing a CFPS system from E. coli. The procedure uses basic lab equipment, a minimal set of reagents, and requires less than one hour to process the bacterial cell mass into a functional S30-T7 extract. BL21(DE3) and MRE600 E. coli strains were used to prepare the S30-T7 extract. The CFPS system was used to produce a set of fluorescent and therapeutic proteins of different molecular weights (up to 66 kDa). This system was able to produce 40–150 μg-protein/ml, with variations depending on the plasmid type, expressed protein and E. coli strain. Interestingly, the BL21-based CFPS exhibited stability and increased activity at 40 and 45°C. To the best of our knowledge, this is the most rapid and affordable lab-scale protocol for preparing a cell-free protein synthesis system, with high thermal stability and efficacy in producing therapeutic proteins.

Antibacterial activity of gold-titanates on Gram-positive cariogenic bacteria

Objective: Gram-positive cariogenic bacteria are etiological agents in dental caries; therefore, strategies to inhibit these bacteria to reduce the incident of this disease have intensified. In this study, we investigated antibacterial activities of titanates and gold-titanates against Lactobacillus casei (Lc) and Streptococcus mutans (Sm).Materials and methods: Monosodium titanate (MST), nanomonosodium titanate (nMST) and amorphous peroxo-titanate (APT), which are inorganic compounds with high-binding affinity for specific metal ions, were used. Total bacterial proteins were measured to represent bacterial cell mass after 24 h incubation with gold-titanates. We further examined the effect of nMST-Au(III) concentrations (10,200,400 mg/L) on Lc and Sm cell viability over time via Live/Dead fluorescent staining and colony forming units (CFUs). Transmission electron microscopy (TEM) was used to determine specific locations on the bacterial cells affected by the nMST-Au(III).Results: We found all gold-titanates and APT alone reduced bacterial protein for Lc (p value <0.001) while only MST-Au(III) and nMST-Au(III) affected Sm growth (p value <0.001). Overall, nMST-Au(III) showed the most effectiveness against both Lc and Sm at 400 mg/L. The Live/Dead staining showed all concentrations of nMST-Au(III) affected Lc growth but only 200 and 400 mg/L nMST-Au(III) interrupted Sm growth. The growth curves based on CFUs/mL showed all nMST-Au(III) concentrations affected growth of both Lc and Sm. TEM images showed nMST-Au(III) attached to Lc and Sm cell wall and were internalized into both cells.Conclusions: nMST-Au(III) demonstrated potential antimicrobial activity against Gram-positive cariogenic bacteria. These results support further development of nMST-Au(III) as a potential novel material to prevent dental caries.