Ionic liquid pretreatment of stinging nettle stems and giant miscanthus for bioethanol production.

Production of ethanol from lignocellulosic biomass is considered the most promising proposition for developing a sustainable and carbon-neutral energy system. The use of renewable raw materials and variability of lignocellulosic feedstock generating hexose and pentose sugars also brings advantages of the most abundant, sustainable and non-food competitive biomass. Great attention is now paid to agricultural wastes and overgrowing plants as an alternative to fast-growing energetic crops. The presented study explores the use of stinging nettle stems, which have not been treated as a source of bioethanol. Apart from being considered a weed, stinging nettle is used in pharmacy or cosmetics, yet its stems are always a non-edible waste. Therefore, the aim was to evaluate the effectiveness of pretreatment using imidazolium- and ammonium-based ionic liquids, enzymatic hydrolysis, fermentation of stinging nettle stems, and comparison of such a process with giant miscanthus. Raw and ionic liquid-pretreated feedstocks of stinging nettle and miscanthus were subjected to compositional analysis and scanning electron microscopy to determine the pretreatment effect. Next, the same conditions of enzymatic hydrolysis and fermentation were applied to both crops to explore the stinging nettle stems potential in the area of bioethanol production. The study showed that the pretreatment of both stinging nettle and miscanthus with imidazolium acetates allowed for increased availability of the critical lignocellulosic fraction. The use of 1-butyl-3-methylimidazolium acetate in the pretreatment of stinging nettle allowed to obtain very high ethanol concentrations of 7.3 g L-1, with 7.0 g L-1 achieved for miscanthus. Results similar for both plants were obtained for 1-ethyl-3-buthylimidazolium acetate. Moreover, in the case of ammonium ionic liquids, even though they have comparable potential to dissolve cellulose, it was impossible to depolymerize lignocellulose and extract lignin. Furthermore, they did not improve the efficiency of the hydrolysis process, which in turn led to low alcohol concentration. Overall, from the presented results, it can be assumed that the stinging nettle stems are a very promising bioenergy crop.

Synergic Effect of Selected Ingredients and Calcium Chloride on the Technological, Molecular and Microbial Usefulness of Eggshells and Their Impact on Sensory Properties in a Food Model System.

Lower levels of calcium in adults increase the risk for osteoporosis, and in children, low calcium levels can impact their potential adult height. The study objective was to analyze the bioavailability and physicochemical properties of a calcium preparation based on chicken eggs. The base calcium preparation was enriched with one of a variety of biologically active substances, inter alia, vitamin D3, vitamin K, lysine, lactose, magnesium chloride and inulin. The newly developed calcium preparations were subjected to structural analysis using X-ray diffraction and scanning electron microscopy, and the hydrodynamic diameter for the molecules was determined using the dynamic light scattering method and their zeta potential. To determine the optimum storage conditions of calcium preparations, their hygroscopicity and bulk density were determined. The calcium preparations were also added to selected food products, such as apple juice with mango, fruit dessert (jelly) and beef meatballs. The enriched food products were subjected to sensory analysis. The study demonstrated the significant influence of additives to calcium preparation in terms of its hygroscopicity and morphology. It was found that all products with the addition of analyzed preparations were characterized by high sensory desirability. The results presented in the study comprise the basis for the development of new food products, enriched with calcium.

Effect of fortification with calcium from eggshells on bioavailability, quality, and rheological characteristics of traditional Polish bread spread.

Hen eggshells are a rich and natural source of calcium and can serve as a biofunctional food ingredient. Enriching the traditional Polish bread spread (sersmazony) with micronized eggshell is an attractive proposition for consumers who require easily available calcium. The present study aimed to evaluate the use of micronized eggshells as a source of bioavailable calcium in bread spread. The study evaluated the effect of selected biocomponents on calcium bioavailability by using an in vitro digestion model. The enrichment of bread spread with eggshell, lysine, vitamin D3, and vitamin K enhanced all examined physicochemical variables except water activity. Enrichment with eggshells increased calcium levels >2.5-fold compared with the control sample. As an ingredient of bread spread, lysine is an important rheological factor. The bioavailability of calcium was higher in samples with lysine and vitamin K compared with samples that contained eggshell alone.

Assessment of wasteland derived biomass for bioethanol production

Background: The bioethanol produced from biomass is a promising alternative fuel. The lignocellulose from marginal areas or wasteland could be a promising raw material for bioethanol production because it is present in large quantities, is cheap, renewable and has favorable environmental properties. Despite these advantages, lignocellulosic biomass is much more difficult to process than cereal grains, due to the need for intensive pretreatment and relatively large amounts of cellulases for efficient hydrolysis. Therefore, there is a need to develop an efficient and cost-effective method for the degradation and fermentation of lignocellulosic biomass to ethanol. Results: The usefulness of lignocellulosic biomass from wasteland for the production of bioethanol using pretreatment with the aid of ionic liquids of 1-ethyl-3-methylimidazolium acetate and 1-ethyl-3-methylimidazolium chloride was evaluated in this study. The pretreatment process, enzymatic hydrolysis and alcoholic fermentation lasted a total of 10 d. The largest amounts of bioethanol were obtained from biomass originating from agricultural wasteland, in which the dominant plant was fireweed (Chamaenerion angustifolium) and from the field where the common broom (Cytisus scoparius) was the dominant. Conclusions: The plants such as fireweed, common broom, hay and goldenrod may be useful for the production of liquid biofuels and it would be necessary in the further stage of research to establish and optimize the conditions for the technology of ethyl alcohol producing from these plant species. Enzymatic hydrolysis of biomass from agricultural wastelands results in a large increase in fermentable sugars, comparable to the enzymatic hydrolysis of rye, wheat, rice or maize straw.

Composition and In Vitro Effects of Cultivars of Humulus lupulus L. Hops on Cholinesterase Activity and Microbial Growth.

Common hop (Humulus lupulus L.) has significant health-promoting properties. Hop cones contain resins, essential oils, proteins, polyphenols, lipids, waxes, and cellulose. Hop extracts include bioactive compounds such as polyphenolic compounds (phenolic acids, and flavonols), and chlorophylls. The aim of this study was to compare the pro-health potential of hop cone extracts obtained from three cultivars (Magnum, Lubelski, and Marynka). The results showed that the cones of Magnum cultivar demonstrated the highest biological activity. The sum of phenolic acids and flavonols in ethanol extract was the highest for this variety and was equal 4903.5 µg/g dw. Ethanol extracts of Magnum cultivars showed the highest degree of iron ion chelation (55.43-88.76%) as well as the activity against 1,1-diphenyl-2-picrylhydrazyl radical (4.75 mmol Tx/g dw). Hop cone extracts as cholinesterase inhibitors showed high potential for aqueous variants. In terms of antimicrobial activity, all investigated extracts demonstrated strong inhibition against Staphylococcus aureus and Staphylococcus epidermidis, with the Magnum cultivar showing the strongest inhibition. Owing to the biofunctional features of hop cone, it can be concluded that it is an attractive raw material with pro-health potential that can be used much more widely in food technology. However, it should be noted that toxicological tests and in vitro tests must be carried out before the raw material is used in food production.

Cyclodextrins as multifunctional excipients: Influence of inclusion into ß-cyclodextrin on physicochemical and biological properties of tebipenem pivoxil.

A novel approach for drug design based on the oral carbapenem analog tebipenem pivoxil (TP) has been proposed. The formation of the tebipenem pivoxil-ß-cyclodextrin (TP-ß-CD) complex resulted in changes concerning physicochemical properties of TP, which is significant for planning the development of an innovative pharmaceutical formulation as well as in the modifications of biological activity profile of the studied delivery system. The inclusion of TP into ß-cyclodextrin (ß-CD) was confirmed by spectral (infrared and Raman spectroscopies) and thermal method (differential scanning calorimetry). Precise indications of TP domains responsible for interaction with ß-CD were possible through a theoretical approach. The most important physicochemical modifications obtained as an effect of TP inclusion were changes in solubility and its rate depending on acceptor fluids, and an increase in chemical stability in the solid state. Biologically essential effects of TP and ß-CD interactions were decreased TP permeability through Caco-2 cell monolayers with the use of efflux effect inhibition and increased antibacterial activity. The proposed approach is an opportunity for development of the treatment in resistant bacterial infections, in which along with physicochemical modifications induced by a drug carrier impact, a carrier synergy with a pharmacological potential of an active pharmaceutical substance could be used.

Enhanced pharmacological efficacy of sumatriptan due to modification of its physicochemical properties by inclusion in selected cyclodextrins.

The study focused on the pharmacological action of sumatriptan, in particular its antiallodynic and antihyperalgesic properties, as an effect of cyclodextrinic inclusion of sumatriptan, resulting in changes of its physicochemical qualities such as dissolution and permeability through artificial biological membranes, which had previously been examined in vitro in a gastro-intestinal model. The inclusion of sumatriptan into ß-cyclodextrin and 2-hydroxylpropylo-ß-cyclodextrin by kneading was confirmed with the use of spectral (fourier-transform infrared spectroscopy (FT-IR); solid state nuclear magnetic resonance spectroscopy with magic angle spinning condition, 1H and 13C MAS NMR) and thermal (differential scanning calorimetry (DSC)) methods. A precise indication of the domains of sumatriptan responsible for its interaction with cyclodextrin cavities was possible due to a theoretical approach to the analysis of experimental spectra. A high-performance liquid chromatography with a diode-array detector method (HPLC-DAD) was employed to determine changes in the concentration of sumatriptan during dissolution and permeability experiments. The inclusion of sumatriptan in complex with cyclodextrins was found to significantly modify its dissolution profiles by increasing the concentration of sumatriptan in complexed form in an acceptor solution compared to in its free form. Following complexation, sumatriptan manifested an enhanced ability to permeate through artificial biological membranes in a gastro-intestinal model for both cyclodextrins at all pH values. As a consequence of the greater permeability of sumatriptan and its increased dissolution from the complexes, an improved pharmacological response was observed when cyclodextrin complexes were applied.

Evaluation of the ability to metabolize 1, 2-propanediol by heterofermentative bacteria of the genus Lactobacillus

Background: New directions of research on lactic acid bacteria include investigation of metabolic pathways for the synthesis and/or metabolism of 1,2-propanediol, commonly used in the food and chemical industry, medicine, pharmacy and cosmetology as well as agriculture. The objective of this study was to compare the capacity of strains representing three diverse heterofermentative species belonging to the genus Lactobacillus to synthesize and/or transform 1,2-PD as well as to suggest new directions of research aimed at commercial use of this metabolite. Results: The novel strain of Lactobacillus buchneri A KKP 2047p, characterized as exhibiting an unusual trait for that species in the form of capacity to metabolize 1,2-PD, grew poorly in a medium containing 1,2-PD as a sole carbon source. The supplementation with glucose facilitated rapid growth of bacteria and use of 1,2-PD for the synthesis of propionic acid. A similar observation was noted for Lactobacillus reuteri. On the other hand, Lactobacillus diolivorans effectively metabolized 1,2-PD which was the sole carbon source in the medium, and the addition of glucose inhibited the synthesis of propionic acid. The experiments also investigated the effect of cobalamin as a diol dehydratase coenzyme involved in the propionic acid synthesis from 1,2-PD whose addition promoted the yield of the reaction in the case of all tested strains. Conclusions: All tested isolates showed the ability to effectively metabolize 1,2-PD (in the presence of cobalamin) and its conversion to propionic acid, which reveals that investigated bacteria meet the essential requirements of microorganisms with a potential application.

Radiolytic studies of cefozopran hydrochloride in the solid state

Background: The radiation sterilization is one of the best methods for sterilizing vulnerable degradation drugs like cefozopran hydrochloride. Results: Chemical stability of radiosterylized cefozopran hydrochloride, was confirmed by spectrophotometric and chromatographic methods. EPR studies showed that radiation has created some radical defects whose concentration was no more than several dozen ppm. The antibacterial activity of cefozopran hydrochloride irradiated with a dose of 25 kGy was unaltered for Gram-positive bacteria but changed for two Gram-negative strains. The radiation sterilized cefozopran hydrochloride was not in vitro cytotoxic against human CCD39Lu normal lung fibroblast cell line. Conclusions: Cefozopran hydrochloride in solid state is not resistant to radiation sterilization and this method cannot be used for sterilization of this compound.

ß-Cyclodextrin complexation as an effective drug delivery system for meropenem.

Following the preparation of an inclusion complex of ß-cyclodextrin and meropenem, methods based on FT-IR, Raman and DSC were used for its characterization. An analysis of changes in the stability of meropenem after complexation showed that the complex may serve as a valuable delivery system significantly contributing to enhanced meropenem stability in aqueous solutions and in the solid phase. Due to a sustained transfer of meropenem from the cavity of the cyclodextrin it was possible to maintain a constant desired meropenem concentration over a period of 20 h, as confirmed by a release study. An evaluation of microbial activity not only demonstrated that the bactericidal action of meropenem was not stopped as a result of complexation but even pointed to greater growth inhibition in certain clinically important strains. The fact that investigations of meropenem stability and microbial activity proposed the carbonyl groups as those domains of a meropenem molecule that are instrumental in the formation of a complex with ß-cyclodextrin supports the findings of theoretical studies based on molecular modeling.

The radiolytic studies of cefpirome sulfate in the solid state.

The possibility of applying radiation sterilization to cefpirome sulfate was investigated. The lack of changes in the chemical structure of cefpirome sulfate irradiated with a dose of 25 kGy, required to attain sterility, was confirmed by UV, FT-IR, Raman, DSC and chromatographic methods. Some radical defects with concentration no more than over a several dozen ppm were created by radiation. The antibacterial activity of cefpirome sulfate for two Gram-positive and three Gram-negative strains was changed. The radiation sterilised cefpirome sulfate was not in vitro cytotoxic against fibroblast cells.

Complex of rutin with ß-cyclodextrin as potential delivery system.

This study aimed to obtain and characterize an RU-ß-CD complex in the context of investigating the possibility of changes in the solubility, stability, antioxidative and microbiological activity as well as permeability of complexated rutin as against its free form. The formation of the RU-ß-CD complex via a co-grinding technique was confirmed by using DSC, SEM, FT-IR and Raman spectroscopy, and its geometry was assessed through molecular modeling. It was found that the stability and solubility of the so-obtained complex were greater compared to the free form; however, a slight decrease was observed inits antibacterial potency. An examination of changes in the EPR spectra of thecomplex excluded any reducing effect of complexation on the antioxidative activity of rutin. Considering the prospect of preformulation studies involving RU-ß-CD complexes, of significance is also the observed possibility of prolongedly releasing rutin from the complex at a constant level over along period of 20 h, and the fact that twice as much complexated rutin was able topermeate compared to its free form.

1,3-Propanediol production by new recombinant Escherichia coli containing genes from pathogenic bacteria.

1,3-Propanediol (1,3-PDO) is an organic compound, which is a valuable intermediate product, widely used as a monomer for synthesizing biodegradable polymers, increasing their strength; as well as an ingredient of textile, cosmetic and medical products. 1,3-PDO is mostly synthesized chemically. Global companies have developed technologies for 1,3-PDO synthesis from petroleum products such as acrolein and ethylene oxide. A potentially viable alternative is offered by biotechnological processes using microorganisms capable of synthesizing 1,3-PDO from renewable substrates (waste glycerol, a by-product of biofuel production, or glucose). In the present study, genes from Citrobacter freundii and Klebsiella pneumoniae were introduced into Escherichia coli bacteria to enable the synthesis of 1,3-PDO from waste glycerol. These strains belong to the best 1,3-PDO producers, but they are pathogenic, which restricts their application in industrial processes. The present study involved the construction of two gene expression constructs, containing a total of six heterologous glycerol catabolism pathway genes from C. freundii ATCC 8090 and K. pneumoniae ATCC 700721. Heterologous genes encoding glycerol dehydratase (dhaBCE) and the glycerol dehydratase reactivation factor (dhaF, dhaG) from C. freundii and gene encoding 1,3-PDO oxidoreductase (dhaT) from K. pneumoniae were expressed in E. coli under the control of the T7lac promoter. An RT-PCR analysis and overexpression confirmed that 1,3-PDO synthesis pathway genes were expressed on the RNA and protein levels. In batch fermentation, recombinant E. coli bacteria used 32.6gl(-1) of glycerol to produce 10.6 gl(-1) of 1,3-PDO, attaining the efficiency of 0.4 (mol1,3-PDO molglycerol(-1)). The recombinant E. coli created is capable of metabolizing glycerol to produce 1,3-PDO, and the efficiency achieved provides a significant research potential of the bacterium. In the face of shortage of fossil fuel supplies and climate warming there is an increasing industrial need to exchange the chemical way of chemicals synthesis for biotechnological - more ecological manner. The 1,3-PDO production from glycerol is an desirable alternative to the traditional production from non-renewable resources. This work is a part of project, which opens a way to development of innovative "green chemistry" and new perspectives to chemical industry.

Stability, compatibility and microbiological activity studies of meropenem-clavulanate potassium.

Meropenem (MEM) and clavulanate potassium have been reported to demonstrate highly effective activity against Mycobacterium tuberculosis. There have been no reports on research into the complex of these chemotherapeutics concerning their mutually dependent stability or microbiological action on other microorganisms. Stability and compatibility studies of MEM/clavulanate potassium were conducted by using an HPLC-DAD method. The antibacterial activity of MEM/clavulanate potassium was tested in vitro against a selection of indicator bacteria strains by determining the MIC as well as analyzing the kinetics of changes in the concentrations of Pseudomonas aeruginosa, Staphylococcus aureus and Listeria monocytogenes caused by the action of MEM/clavulanate potassium. The stability and compatibility of MEM/clavulanate potassium were examined in aqua pro iniectione, 0.9% NaCl and 5% glucose at room temperature and at 5 °C. The degradation rates of MEM/clavulanate potassium depended on the type of infusion solvent used. Although in aqueous solutions of MEM/clavulanate potassium neither compound showed any mutual impact on the rate of degradation, clavulanate potassium was more labile than MEM. The synergy between these two resulted in a significantly lower value of MIC as compared to the values observed for the individual activity of either compound. The infusion solvent in which compatibility is observed between the components of the mixture MEM/clavulanate potassium is aqua pro iniectione. The complex MEM/clavulanate potassium demonstrates synergic antibacterial activity against P. aeruginosa, S. aureus and L. monocytogenes.

Effect of 1,3-propanediol, organic acids, and ethanol on growth and metabolism of Clostridium butyricum DSP1.

Knowledge of tolerance of bacteria to toxic stress is important, especially for processes targeted at high final titers of product. Information on environmental limits and stress responses may help during selection of strains or design and control of processes. The influence of the main product and its co-products on the process of 1,3-propanediol (PD) synthesis was determined. Adaptation to toxic compounds was noticed as Clostridium butyricum DSP1 was less sensitive to the addition of these factors during its exponential growth on glycerol than when the factor was present in the medium before inoculation. It was also shown that the response of the tested strain to the toxicity of 1,3-propanediol (1,3-PD) has different proteomic profiles depending on the stage of culture when this substance is introduced. Relatively satisfactory activity of the analyzed strain was sustained up to a concentration of 1,3-PD of 40 g/L while 80 g/L of this metabolite was lethal to the bacterium. As for the by-products, acetic acid was determined to be the most toxic among the acids excreted during the process.

Biotechnological potential of Clostridium butyricum bacteria.

In response to demand from industry for microorganisms with auspicious biotechnological potential, a worldwide interest has developed in bacteria and fungi isolation. Microorganisms of interesting metabolic properties include non-pathogenic bacteria of the genus Clostridium, particularly C. acetobutylicum, C. butyricum and C. pasteurianum. A well-known property of C. butyricum is their ability to produce butyric acid, as well as effectively convert glycerol to 1,3-propanediol (38.2 g/L). A conversion rate of 0.66 mol 1,3-propanediol/mol of glycerol has been obtained. Results of the studies described in the present paper broaden our knowledge of characteristic features of C. butyricum specific isolates in terms of their phylogenetic affiliation, fermentation capacity and antibacterial properties.

1, 3-Propanediol production from crude glycerol by Clostridium butyricum DSP1 in repeated batch

Background The production of biofuels from renewable energy sources is one of the most important issues in industrial biotechnology today. The process is known to generate various by-products, for example crude glycerol, which is obtained in the making of biodiesel from rapeseed oil. Crude glycerol may be utilized in many ways, including microbial conversion to 1,3-propanediol (1,3-PD), a raw material for the synthesis of polyesters and polyurethanes. Results The paper presents results of a study on the synthesis of 1,3-propanediol from crude glycerol by a repeated batch method with the use of Clostridium butyricum DSP1. Three cycles of fermentation medium replacement were carried out. The final concentration of 1,3-PD was 62 g/L and the maximum productivity, obtained during the second cycle, reached 1.68 g/L/h. Additionally, experiments conducted in parallel to the above involved using the entire quantity of the culture broth removed from the bioreactor to inoculate successive portions of fermentation media containing crude glycerol at concentrations of 80 g/L and 100 g/L. Under those conditions, the maximum 1,3-PD concentrations were 43.2 g/L and 54.2 g/L. Conclusions The experiments proved that by using a portion of metabolically active biomass as inoculum for another fermentation formula it is possible to eliminate the stage of inoculum growth and thereby reduce the length of the whole operation. Additionally, that strategy avoids the phase of microbial adaptation to a different source of carbon such as crude glycerol, which is more difficult to utilize, thus improving the kinetic parameters of 1,3-PD production.

Kinetic modeling of simultaneous saccharification and fermentation of corn starch for ethanol production.

Fuel ethanol production, using a simultaneous saccharification and fermentation process (SSF) of native starch from corn flour, has been performed using Saccharomyces cerevisiae and a granular starch hydrolyzing enzyme. The quantitative effects of mash concentration, enzyme dose and pH were investigated with the use of a Box-Wilson central composite design protocol. Proceeding from results obtained in optimal fermentation conditions, a kinetics model relating the utilization rates of starch and glucose as well as the production rates of ethanol and biomass was tested. Moreover, scanning electron microscopy (SEM) was applied to investigate corn starch granule surface after the SFF process. A maximum ethanol concentration of 110.36 g/l was obtained for native corn starch using a mash concentration of 25%, which resulted in ethanol yield of 85.71%. The optimal conditions for the above yield were found with an enzyme dose of 2.05 ml/kg and pH of 5.0. These results indicate that by using a central composite design, it is possible to determine optimal values of the fermentation parameters for maximum ethanol production. The investigated kinetics model can be used to describe SSF process conducted with granular starch hydrolyzing enzymes. The SEM micrographs reveal randomly distributed holes on the surface of granules.

An increasing of the efficiency of microbiological synthesis of 1, 3-propanediol from crude glycerol by the concentration of biomass

Background 1,3-Propanodiol (1,3-PD), is used in the production of polytrimethylene terephthalate (PTT), an aromatic polyester that exhibits high elastic recoveries. It is also employed as a supplement with low solidification properties, a solvent and a lubricant in the formof propylene glycol. 1,3-PD is effectively synthesized by a microbiological way from crude glycerol. The main problem of this technology is using a high concentration of glycerol, which is a limiting factor for bacteria cells growth (especially in batch fermentation). Results In this work, the influence of different glycerol concentration in batch fermentation on Clostridium butyricum DSP1 metabolism was investigated. The biomass was concentrated for two times with the use of membrane module (in case of increasing kinetic parameters). Increased optical density of bacteria cells six times increased the productivity of 1,3-PD in cultivation with 20 g/L of glycerol at the beginning of the process, and more than two times in cultivation with 60-80 g/L. Also the possibility of complete attenuation of 140 g/L of crude glycerol in the batch fermentation was investigated. During the cultivation, changes of protein profiles were analyzed. The most significant changes were observed in the cultivation in the medium supplemented with 80 g/L of glycerol. They related mainly to the DNA protein reconstructive systems, protective proteins (HSP), and also the enzymatic catalysts connected with glycerol metabolic pathway. Conclusions The application of filtration module in batch fermentation of crude glycerol by C. butyricum DSP1 significantly increased the productivity of the process.