Cell Biomass Research Articles

Sequestration of cobalt and nickel by biofilm forming bacteria isolated from spent nuclear fuel pool water

In the current study, six bacterial types, isolated from spent nuclear fuel (SNF) pool facility, were investigated for their ability to sequester heavy metals (cobalt and nickel). Biofilm formation by the six bacterial isolates, viz., Bacillus subtilis, Staphylococcus species, Staphylococcus arlettae, Staphylococcus epidermidis, Staphylococcus auricularis, and Chryseobacterium gleum, were assayed, and they were found to have significant biofilm forming property. Their biofilms were characterised using confocal scanning laser microscopy, and their potential to accumulate Co2+ and Ni2+ from bulk solutions was analysed with respect to time. A comparative assessment of bioaccumulation capacity was done using biofilms, planktonic cells, and live vs dead cells. The strains accumulated Co2+ and Ni2+ in the range of 4 × 10-4 to 1 × 10-5g/mg of cell biomass. It is interesting to note that dead biomass also showed significant removal of the two metal ions, suggesting an alternative process for metal removal. This study suggests that hostile environments can be a repertoire of putative bacterial species with potential heavy metals and other contaminants remediation properties.

Biomass and lipid production by the native green microalgae Chlorella sorokiniana in response to nutrients, light intensity, and carbon dioxide: experimental and modeling approach.

Introduction: Microalgae are photosynthetic cells that can produce third-generation biofuels and other commercial compounds. Microalgal growth is influenced by two main parameters: light intensity and carbon dioxide concentration, which represent the energy and carbon source, respectively. For photosynthesis, the optimum values of abiotic factors vary among species. Methods: In this study, the microalga Chlorella sorokiniana was isolated from a freshwater lake. It was identified using molecular analysis of the ribosomal internal transcribed spacer. A single-factor design of experiments in 250-mL Erlenmeyer flasks was used to evaluate which concentrations of nitrogen and phosphorus increase the production of biomass and lipids. The response surface methodology was used with a 32-factorial design (light intensity and CO2 were used to evaluate its effect on biomass, lipid production, and specific growth rates, in 200-mL tubular photobioreactors (PBRs)). Results and Discussion: Low levels of light lead to lipid accumulation, while higher levels of light lead to the synthesis of cell biomass. The highest biomass and lipid production were 0.705 ± 0.04g/L and 55.1% ± 4.1%, respectively. A mathematical model was proposed in order to describe the main phenomena occurring in the culture, such as oxygen and CO2 mass transfer and the effect of light and nutrients on the growth of microalgae. The main novelties of this work were molecular identification of the strain, optimization of culture conditions for the indigenous microalgae species that were isolated, and formulation of a model that describes the behavior of the culture.

A marine lipopeptides-producing Bacillus amyloliquefaciens HY2-1 with a broad-spectrum antifungal and antibacterial activity and its fermentation kinetics study.

The antagonistic Bacillus amyloliquefaciens HY2-1 was a marine microbiology that was isolated previously from the seabed silt of Beibu Gulf in China by dual culture with Penicillium digitatum. As a continuous study, the present work focused on evaluating the antimicrobial activity, identifying the produced active components, and revealing the fermentation characteristics of B. amyloliquefaciens HY2-1, respectively. It was found that B. amyloliquefaciens HY2-1 exhibited a broad-spectrum antimicrobial activity against the tested seven phytopathogenic fungi and five pathogenic bacteria by producing Bacillus lipopeptides such as fengycin A (C14 to C19 homologues) and surfactin (C14 and C15 homologues). Morphological observation of P. digitatum under light microscope, scanning electron microscopy, transmission electron microscopy, and fluorescence microscope inferred that B. amyloliquefaciens exerted the antagonistic activity by damaging the fungal cell membrane, thus inhibiting the mycelium growth and sporification of phytopathogenic fungi. As a marine microbiology, our results showed that B. amyloliquefaciens could survive and metabolize even at the culture condition with 110g/L of NaCl concentration, and the produced antimicrobial compounds exhibited excellent thermostability and acid-alkali tolerance. The dynamic models were further constructed to theoretically analyze the fermentation process of B. amyloliquefaciens HY2-1, suggesting that the synthesis of antimicrobial compounds was coupled with both cell growth and cell biomass. In conclusion, the marine lipopeptides-producing B. amyloliquefaciens HY2-1 showed a promising prospect to be explored as a biocontrol agent for plant disease control of crops and postharvest preservation of fruits and vegetables, especially due to its outstanding stress resistance and the broad-spectrum and effective antagonist on various phytopathogenic fungi.

Evaluation of the Amino Acid Composition and Content of Organic Acids of Complex Postbiotic Substances Obtained on the Basis of Metabolites of Probiotic Bacteria Lacticaseibacillus paracasei ABK and Lactobacillus helveticus H9

In this article, the probiotic strains of Lacticaseibacillus paracasei ABK and Lactobacillus helveticus H9 were cultured in reconstituted skim milk (RSM medium) and MRS broth, and the cell biomass was removed at the end of fermentation in order to obtain postbiotic substances. In postbiotics, the composition of total amino acids, and also the content of free amino acids and organic acids were analyzed. It was shown that in all RSM-based postbiotic substances the concentration of all free amino acids increased. On the contrary, in the MRS-based postbiotics free amino acids were mostly consumed during fermentation; however, a substantial, two-fold, decrease in methionine concentration was observed in postbiotics obtained with L. paracasei ABK. Both L. paracasei ABK and L. helveticus H9 strains showed change in their fermentation profile from homofermentative in MRS broth to mix-acid fermentation in RSM medium. Both strains produced lactic acid in the investigated media and produced lactate together with acetate in RSM. L. helveticus H9 additionally synthesizes succinic acid on both media. Thus, it has been shown that RSM is more preferable than MRS for fermentation with L. paracasei ABK and L. helveticus H9 for obtaining postbiotics enriched with free amino acids and organic acids.

Sequential extraction of compounds of interest from yeast biomass assisted by pulsed electric fields.

One strategy to reduce cost and improve feasibility of waste-yeast biomass valorization is to obtain a spectrum of marketable products rather than just a single one. This study explores the potential of Pulsed Electric Fields (PEF) for the development of a cascade process designed to obtain several valuable products from Saccharomyces cerevisiae yeast biomass. Yeast biomass was treated by PEF, which affected the viability of 50%, 90%, and over 99% of S. cerevisiae cells, depending on treatment intensity. Electroporation caused by PEF allowed access to the cytoplasm of the yeast cell without causing total breakdown of the cell structure. This outcome was an essential prerequisite to be able to perform a sequential extraction of several value-added biomolecules from yeast cells located in the cytosol and in the cell wall. After incubating yeast biomass previously subjected to a PEF treatment that affected the viability of 90% of cells for 24h, an extract with 114.91 ± 2.86, 7.08 ± 0.64, and 187.82 ± 3.75mg/g dry weight of amino acids, glutathione, and protein, respectively, was obtained. In a second step, the extract rich in cytosol components was removed after 24h of incubation and the remaining cell biomass was re-suspended with the aim of inducing cell wall autolysis processes triggered by the PEF treatment. After 11days of incubation, a soluble extract containing mannoproteins and pellets rich in β-glucans were obtained. In conclusion, this study proved that electroporation triggered by PEF permitted the development of a cascade procedure designed to obtain a spectrum of valuable biomolecules from S. cerevisiae yeast biomass while reducing the generation of waste.

Precursors and elicitor induced enhancement of cell biomass and phenolic compounds in cell suspensions of Indian basil-Ocimum basilicum (CIM-Saumya).

The online version contains supplementary material available at 10.1007/s12298-023-01316-6.

Synbiotics production using Lactobacillus reuteri EC01, a strain that produces alternan-type exopolysaccharide

Alternan is an α-glucan polysaccharide with a unique structure that has promise for use as a prebiotic. In this study, we isolated a novel strain of Lactobacillus reuteri BR120 that produces abundant exopolysaccharides (EPSs). Monosaccharide analysis and NMR spectroscopy confirmed the major EPS had an alternan-type structure (α-D-glucan with α-1,6 and α-1,3 linkages), with an average molecular weight of 9.3 × 106 Da. L. reuteri EC01, obtained through gamma-ray mutagenesis of L. reuteri BR120, had a 1.3-fold increased production of alternan-type compound. The prebiotic effect of the alternan-type EPS was similar to that of fructans, metabolized by strains of Bifidobacterium longum and B. breve. Additionally, L. reuteri EC01 had various probiotic activities, with anti-inflammatory, radical scavenging, acid tolerance, bile tolerance, and intestinal adhesion effects. L. reuteri EC01 produced 54.3 g/L of the alternan-type EPS through fed-batch fermentation without aeration at 37 °C, 50 rpm, and pH 5.5. The productivity was 0.91 g/L/h, and the conversion yield was 0.279 g alternan-type EPS/g sucrose. This report describes the highest reported production of an alternan-type EPS from fermentation of Lactobacillus spp. The results suggest the cell biomass and EPS derived from fermentation of L. reuteri EC01 have potential use as synbiotics.

Biodiesel production from a novel renewable source through the ultrasound-assisted transesterification process, using energy efficient nanocatalyst developed from waste material

The depletion of fossil fuels created a thrust for an alternative renewable energy source, which could involve sustainable energy formation and cost-reducing catalysts. The catalyst derived from the waste material, specifically a heterogenous nanocatalyst, could economically add value to the process. In this study, a novel microbial strain, Rhodotorula mucilaginosa strain SML (RM-SML), was used as a source of oil for biodiesel production. The nanocatalyst developed from the Central Avian Research Institute – Broiler (CARIB) breed eggshell was used as a catalyst for transesterification, increasing the sustainability and economic viability of the process. RM-SML yielded 68.79 % of oil to its total dry cell biomass. The calcium carbonate-rich CARIB breed eggshell was calcinated in this study to synthesize a highly dynamic heterogeneous CARIB breed eggshell nanocatalyst (CBESNC). The calcination process parameters involved in the nanocatalyst synthesis were optimized using response surface methodology, yielding a maximum calcium oxide of 38.827 %. The synthesized CBESNC was characterized for its size by scanning electron microscopy imaging and its crystallographic nature by X-ray diffraction analysis. CBESNC was used as a heterogeneous catalyst in the ultrasonic-assisted transesterification of RM-SML bio-oil. The effect of the CBESNC in RM-SML bio-oil transesterification was studied and optimized using the response surface methodology, which yielded a maximum biodiesel conversion of 98.33 %. The prediction model in optimization studies was found to fit with the process and was validated experimentally. RM-SML biodiesel was studied for its physicochemical properties and found to meet American Society for Testing and Materials biodiesel standards. It was further confirmed with Gas Chromatography-Mass Spectrum analysis. The energy analysis results proved the commercialization feasibility of the study.

Kinetics of NK-92 growth and functionality in pseudo-static cultures

Understanding the growth and functionality variation of lymphocytes during expansion is fundamental for process control and endpoint determination. However, monitoring CQAs along lymphocyte expansion requires offline cell characterization, hindering timely identification of process deviations and transitions. We developed a culturing protocol that allowed to investigate day-to-day variation of NK-92 cells’ cytotoxicity against K-562 cells. Replacing 20% of the culture with fresh medium every day while disrupting the formed lymphocyte aggregates, morphological and functionality changes of the NK-92 cells were induced. Our experiments revealed that the adaptation to the pseudo-static protocol divided the culture to two distinctive phases with exponential growth in each. In between, there was an intermittent drop in viability. Significant functionality fluctuations were observed, despite the regular stimulation strategy. Scale-independent descriptors, such as growth rate, were calculated for each phase and the whole culture and correlated with cell morphology parameters and functionality. A novel correlation was found between, the cell size-derived, biomass volume and cytotoxicity that was stronger during the first phase, but it was apparent throughout the entire culture. This was possible to capture, only because of the day-to-day monitoring of cytotoxicity. By utilizing the pseudo-static culturing protocol and the methodology, variables, such as biomass volume, were discovered and by this, we are getting closer to find performance indicators suitable to monitor CQAs in a straightforward, non-invasive manner.

Suppression of ETI by PTI priming to balance plant growth and defense through an MPK3/MPK6-WRKYs-PP2Cs module

Pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) are required for host defense against pathogens. Although PTI and ETI are intimately connected, the underlying molecular mechanisms remain elusive. In this study, we demonstrate that flg22 priming attenuates Pseudomonas syringae pv. tomato DC3000 (Pst) AvrRpt2-induced hypersensitive cell death, resistance, and biomass reduction in Arabidopsis. Mitogen-activated protein kinases (MAPKs) are key signaling regulators of PTI and ETI. The absence of MPK3 and MPK6 significantly reduces pre-PTI-mediated ETI suppression (PES). We found that MPK3/MPK6 interact with and phosphorylate the downstream transcription factor WRKY18, which regulates the expression of AP2C1 and PP2C5, two genes encoding protein phosphatases. Furthermore, we observed that the PTI-suppressed ETI-triggered cell death, MAPK activation, and growth retardation are significantly attenuated in wrky18/40/60 and ap2c1 pp2c5 mutants. Taken together, our results suggest that the MPK3/MPK6-WRKYs-PP2Cs module underlies PES and is essential for the maintenance of plant fitness during ETI.

PHAs production by facultative anaerobic bacteria Bacillus cereus FM5 through submerged and solid-state fermentation under anoxic condition.

PHAs (polyhydroxyalkanoates) are the bio-polyester synthesized by different aerobic and anaerobic bacteria as energy storage granule. However, its synthesis by anaerobes or facultative anaerobes is an imperative part of their physiology via assimilating broad range of substrates than aerobes. Thus, three Gram positive facultative anaerobic PHAs producers viz., Enterococcus sp. FM3, Actinomyces sp. CM4 and Bacillus sp. FM5 were selected. Among them, Bacillus sp. FM5 showed higher cell biomass production in MSM (mineral salt medium) comprised of glucose & peptone as carbon & nitrogen source at pH 9, temperature 37°C, inoculum 10% and incubation period 72h. Under optimized condition, Bacillus sp. FM5 produced 0.89 and 1.5gl-1 of PHAs through submerged and solid-state fermentation in anoxic condition. In-silico analysis confirmed the facultative anaerobic PHAs producing bacteria as Bacillus cereus FM5. IR spectra of PHAs illustrated a strong absorption peak at 1718.50cm-1 representing carbonyl ester (C=O) functional group of PHB (polyhydroxybutyrate), belonging to the family PHAs. It is the first report demonstrating PHAs production by Bacillus cereus FM5 in anoxic condition through different bioprocess technology, which may pave the way in the arena of further biopolymer research.

Comparison of biofloc formation from microalgal cultivation by auto- and bacteria-associated types of flocculation

Microalgal harvesting has still been a challenge to investigators who take their investiment into microalgal production. The cost for microalgal downstream processing is as high as 20% of the total production of biodiesel. Among hundreds of curent methods of biomass harvesting, autoflocculation and bacteria-based aggregation are still being researched and applied in large-scale producuion. This study implemented a comparison of how microalgal cells aggregate large bioflocs according to two types of flocculation. The microalgal autoflocculation was implemented by adding Ca2+ or Mg2+ with an increase of pH to 11, resulting in cell biomass of 13.7 or 15.5 mg/l, respectively. Meanwhile, the bioflocculation under the support of Bacillus subtilis MT300405 and Escherichia coli ATCC 85922 could produce large bioflocs with a cell biomass of 1.5 times higher than the autoflocculation case without the influence of pH. Moreover, images from scanning electron microscopy indicated differences between two types of flocculation. With the presence of bacteria, microalgal cells were more tightly bound by a membrane or a layer of extracellular polimeric substance, inducing to form large bioflocs. This was not found under autoflocculation.

In-line monitoring of Bordetella pertussis cultivation using fluorescence spectroscopy.

Fluorescence spectroscopy is a non-invasive and highly sensitive method for bioprocess monitoring. The use of fluorescence spectroscopy is not very well established in the industry for in-line monitoring. In the present work, a 2-D fluorometer with two excitation lights (365 and 405 nm) and emission spectra in the range of 350-850 nm were used for in-line monitoring of two strains of Bordetella pertussis cultivation operated in batch and fed batch. A Partial Least Squares (PLS) based regression model was used for the estimation of cell biomass, amino acids (glutamate and proline) and antigen (Pertactin)produced. It was observed that accurate predictions were achieved when models were calibrated separately for each cell strain and nutrient media formulation. Also, prediction accuracy was improved when dissolved oxygen, agitation and culture volume are added as additional features in the regression model. The proposed approach of combining in-line fluorescence and other online measurements is shown to have good potential for in-line monitoring of bioprocesses.

Disruption of Yeast Cells Xanthophyllomyces Dendrorhous (Phaffia Rhodozyma) by Vibration Resonant Low-Frequency Cavitator

The goal of the study is to optimize the mode of disruption of the yeast Phaffia rhodozyma KNH 1 by a vibration-resonant low-frequency cavitator (VLC). The destruction of the cell biomass of yeast culture P. rhodozyma strain KNH 1 was carried out in VLC with water cooling, the capacity of 800 W, and resonant frequencies of vibrations of 30 Hz, 35 Hz, 37 Hz, 37.8 Hz, 39 Hz, 50 Hz, and in the presence of nitrogen in the reaction medium. Our data suggest that the yield of processed biomass by the treatment of yeast culture in VLC depends on the culture age and the mode of the treatment. Thus, for the six-day culture, we got the highest yield by its processing in VLC at 35 Hz for 75 min. The highest yield from the five-day culture was obtained after the treatment in VLC for 1 h at 37-37.8 Hz. The lowest yield of the disrupted yeast cells was obtained after 5 h of treatment in VLC at 37.8 Hz. The high level of yeast cell disruption can be used for the preparation of glucans aqueous solutions. Our data show that for such a level of disruption to treat five-day culture of P. rhodozyma in VLC at 37 Hz resonance frequency with nitrogen gas, bubbling through the reaction medium is economically profitable. For the first time, this study demonstrates the established optimal mode of destruction of yeast cells of P. rhodozyma strain KNH1 for the action of the vibration-resonance low-frequency cavitator or VLC. Ana¬lysis of the presented data indicates that the claimed me¬thod is convenient, efficient, and technologically justified.

Urea as a source of nitrogen and carbon leads to increased photosynthesis rates in Chlamydomonas reinhardtii under mixotrophy

Microalgae is a potential source of bioproducts, including feedstock to biofuels. Urea has been pointed as potential N source for microalgae growth. Considering that urea metabolism releases HCO3- to the medium, we tested the hypothesis that this carbon source could improve photosynthesis and consequently growth rates of Chlamydomonas reinhardtii. In this sense, the metabolic responses of C. reinhardtii grown with ammonium and urea as nitrogen sources under mixotrophic and autotrophic conditions were investigated. Overall, the mixotrophy led to increased cell growth as well as to a higher accumulation of lipids independent of N source, followed by a decrease in photosynthesis over the growth phases. In mixotrophy, urea stimulates growth in terms of cell number and dry weight. Furthermore, higher photosynthesis was verified in late logarithmic phase compared to ammonium. Under autotrophy conditions, although cell number and biomass were reduced, there was higher production of starch independent of N source. Nonetheless, urea-based autotrophic treatments stimulated biomass production compared to ammonium-based treatment. Under mixotrophy higher input of carbon into the cell from acetate and urea optimized photosynthesis and consequently promoted cell growth. Together, these results suggest urea as alternative source of carbon, improving photosynthesis and cell growth in C. reinhardtii.

Modified Activated Carbon: A Supporting Material for Improving Clostridium beijerinckii TISTR1461 Immobilized Fermentation.

This study aimed to investigate the effect of activated carbon (AC) as an immobilization material in acetone-butanol-ethanol fermentation. The AC surface was modified with different physical (orbital shaking and refluxing) and chemical (nitric acid, sodium hydroxide and, (3-aminopropyl)triethoxysilane (APTES)) treatments to enhance the biobutanol production by Clostridium beijerinckii TISTR1461. The effect of surface modification on AC was evaluated using Fourier-transform infrared spectroscopy, field emission scanning electron microscopy, surface area analyses, and X-ray photoelectron spectroscopy, while the fermented broth was examined by high-performance liquid chromatography. The chemical functionalization significantly modified the physicochemical properties of the different treated ACs and further enhanced the butanol production. The AC treated with APTES under refluxing provided the best fermentation results at 10.93 g/L of butanol, 0.23 g/g of yield, and 0.15 g/L/h of productivity, which were 1.8-, 1.5-, and 3.0-fold higher, respectively, than that in the free-cell fermentation. The obtained dried cell biomass also revealed that the treatment improved the AC surface for cell immobilization. This study demonstrated and emphasized the importance of surface properties to cell immobilization.

Production of Secondary Metabolites from Cell Cultures of Sageretia thea (Osbeck) M.C. Johnst. Using Balloon-Type Bubble Bioreactors.

Sageretia thea is used in the preparation of herbal medicine in China and Korea; this plant is rich in various bioactive compounds, including phenolics and flavonoids. The objective of the current study was to enhance the production of phenolic compounds in plant cell suspension cultures of Sageretia thea. Optimum callus was induced from cotyledon explants on MS medium containing 2,4-dichlorophenoxyacetic acid (2,4-D; 0.5 mg L-1), naphthalene acetic acid (NAA, 0.5 mg L-1), kinetin (KN; 0.1 mg L-1) and sucrose (30 g L-1). Browning of callus was successfully avoided by using 200 mg L-1 ascorbic acid in the callus cultures. The elicitor effect of methyl jasmonate (MeJA), salicylic acid (SA), and sodium nitroprusside (SNP) was studied in cell suspension cultures, and the addition of 200 µM MeJA was found suitable for elicitation of phenolic accumulation in the cultured cells. Phenolic and flavonoid content and antioxidant activity were determined using 2,2 Diphenyl 1 picrylhydrazyl (DPPH), 2,2'-azino-bis (3-ethybenzothiazoline-6-sulphonic acid (ABTS), ferric reducing antioxidant power (FRAP) assays and results showed that cell cultures possessed highest phenolic and flavonoid content as well as highest DPPH, ABTS, and FRAP activities. Cell suspension cultures were established using 5 L capacity balloon-type bubble bioreactors using 2 L of MS medium 30 g L-1 sucrose and 0.5 mg L-1 2,4-D, 0.5 mg L-1 NAA, and 0.1 mg L-1 KN. The optimum yield of 230.81 g of fresh biomass and 16.48 g of dry biomass was evident after four weeks of cultures. High-pressure liquid chromatography (HPLC) analysis showed the cell biomass produced in bioreactors possessed higher concentrations of catechin hydrate, chlorogenic acid, naringenin, and other phenolic compounds.

Concomitant strategy of wastewater treatment and biodiesel production using innate yeast cell (Rhodotorula mucilaginosa) from food industry sewerage and its energy system analysis

In many recent studies, the wastewater with high organic matter and high utilization value compounds was treated using yeast. It decreases the effluent contaminants and makes it applicable for reuse in many food industries. This present study focuses on the combined approach of treatment and biorefinery to increase the circular economy in food industries. The two primary goals of the work were to screen the potential oleaginous yeast from the food industry effluent (FIE) collected from the food flavourant producing industry and used for FIE remediation through bioaugmentation and to extract microbial oil of yeast biomass harvested after remediation. Further, the extracted microbial oil was trans-esterified to produce biodiesel. The process of transesterification was optimized for its influencing parameters. After 10 days of treatment of FIE with the yeast cell, the treatment efficiency was analyzed and found to be precise with the typical discharge value. The overall lipid content of the oleaginous yeast (Rhodotorula mucilaginosa SML) used for the FIE treatment was 67.95 w/w% of dry cell biomass. The extracted microbial oil was used for transesterification; the process was optimized through the one-variable analysis approach and response surface methodology optimization using a central composite design. The transesterification process showed maximum conversion (98%) at oil to methanol ratio - 5.0, catalyst concentration - 2.8% and time - 1.15 h. The fatty acid composition and the physicochemical characteristics were compatible with petroleum diesel, making it applicable for alternative biofuel production. Thus, this concomitant strategy has proved efficient for reducing contaminants in FIE and suggested a new sustainable source for biodiesel production. The exergy, energy and mass balance analysis of the biodiesel conversion process proved that this process is the most economically viable one to increase the circular economy of food industries.

Promoting cell growth for bio-chemicals production via boosting the synthesis of L/D-alanine and D-alanyl-D-alanine in Bacillus licheniformis.

Metabolic engineering is a substantial approach for escalating the production of biochemical products. Cell biomass is lowered by system constraints and toxication carried on by the aggregation of metabolites that serve as inhibitors of product synthesis. In order to increase the production of biochemical products, it is important to trace the relationship between alanine metabolism and biomass. According to our investigation, the appropriate concentration of additional L/D-alanine (0.1g/L) raised the cell biomass (OD600) in Bacillus licheniformis in contrast to the control strain. Remarkably, it was also determined that high levels of intracellular L/D-alanine and D-alanyl-D-alanine were induced by the overexpression of the ald, dal, and ddl genes to accelerate cell proliferation. Our findings clearly revealed that 0.2g/L of L-alanine and D-alanine substantially elevated the titer of poly-γ-glutamic acid (γ-PGA) by 14.89% and 6.19%, correspondingly. And the levels of γ-PGA titer were hastened by the overexpression of the ald, dal, and ddl genes by 19.72%, 15.91%, and 16.64%, respectively. Furthermore, overexpression of ald, dal, and ddl genes decreased the by-products (acetoin, 2,3-butanediol, acetic acid and lactic acid) formation by about 14.10%, 8.77%, and 8.84% for augmenting the γ-PGA production. Our results also demonstrated that overexpression of ald gene amplified the production of lichenysin, pulcherrimin and nattokinase by about 18.71%, 19.82% and 21.49%, respectively. This work delineated the importance of the L/D-alanine and D-alanyl-D-alanine synthesis to the cell growth and the high production of bio-products, and provided an effective strategy for producing bio-products.

Antibacterial Efficacy of Irrigants with Varying Osmolarity on E. faecalis Biofilm: An In Vitro Study.

To evaluate the role of the addition of different concentrations of sodium chloride salt to conventional intracanal irrigants to vary their osmotic values and thereby compare their antibacterial efficacy. In an active attachment biofilm model, Enterococcus faecalis (ATCC 29212) biofilms were grown. Sodium chloride salts were added to 100 mL of distilled water to make 6M (hyperosmotic), 0.5M, and 0.25M (hypoosmotic) sodium chloride solutions, respectively. The experimental groups were divided into three groups: Group I: 5.25% sodium hypochlorite, group II: 2% chlorhexidine, and group III: 2% povidone iodine, and four subgroups within these three groups, such as subgroup A (without salt solution), subgroup B (with 6M of hyperosmotic salt solution), subgroup C (with 0.5M of hypoosmotic salt solution), and subgroup D (with 0.25M of hypoosmotic salt solution), respectively. Biofilms were treated with all the subgroups for a contact time of 15 min. A crystal violet assay was done to estimate the bacterial cell biomass. The results revealed that subgroups IIIB, IB, and IID, ID had a statistical reduction in bacterial biomass at p < 0.05. There were no significant differences between subgroups IC, IIC, and IIIC and subgroups IA, IIA, and IIIA. The antibacterial efficacy of all three irrigants was significantly affected by varying the osmolarities. The results prove that the hyperosmotic and hypoosmotic salt solutions, along with irrigants, have enhanced antibacterial efficacy on E. faecalis biofilm due to its ability to vary the turgor pressure of cell wall, as well as the inherent properties of the irrigants such as hypochlorous acid formation, ionic interaction, and free radical interactions.