Sanitization of Biomass in Agricultural Biogas Plants Depends on the Type of Substrates.

Large-scale pig farming is associated with the production of large amounts of animal excrement, which, after processing into the form of, e.g., slurry, are managed on agricultural land as natural fertilizers. The utilization of pig manure on agricultural land in an excessive and uncontrolled manner may pose a threat to zoonoses due to the significant amounts of potentially pathogenic microorganisms within its content. This study aims to determine the impact of the methane fermentation process carried out in two agricultural biogas plants on the efficiency of sanitization of pig slurry, input biomass, and digestate. The biogas plants differed in terms of the substrate used; one used pig slurry from a maternal (breeding) farm (BP-M), and the other utilized pig slurry from a fattening farm (BP-F). The physicochemical analyses showed that the slurry, input biomass, and digestate from the BP-F were characterized by a significantly higher contents of organic dry matter, ash, and ammonium nitrogen than the slurry, input biomass, and digestate from the BP-M. The parameters of the methane fermentation process, including temperature and pH, reached higher values in the BP-F compared to the BP-M. The microbiological analyses led to the conclusion that the efficiency of sanitization of input biomass, including pig slurry, was significantly higher in the BP-F compared to the BP-M. Due to the above findings, locating biogas plants near pig fattening farms should be recommended.

Argon plasma-modified bacterial cellulose filters for protection against respiratory pathogens.

In this work, we present novel, sustainable filters based on bacterial cellulose (BC) functionalized with low-pressure argon plasma (LPP-Ar). The "green" production process involved BC biosynthesis by Komagataeibacter xylinus, followed by simple purification, homogenization, lyophilization, and finally LPP-Ar treatment. The obtained LPP-Ar-functionalized BC-based material (LPP-Ar-BC-bM) showed excellent antimicrobial and antiviral properties against both Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria, and an enveloped bacteriophage phage Φ6, with no cytotoxicity versus murine fibroblasts in vitro. Further, filters consisting of three layers of LPP-Ar-BC-bM had >99 % bacterial and viral filtration efficiency, while maintaining sufficiently low airflow resistance (6 mbar at an airflow of 95 L/min). Finally, as a proof-of-concept, we were able to prepare 80 masks with LPP-Ar-BC-bM filter and ~85 % of volunteer medical staff assessed them as "good" or "very good" in terms of comfort. We conclude that our novel sustainable, biobased, biodegradable filters are suitable for respiratory personal protective equipment (PPE), such as surgical masks and respirators.

The cross-linked bacterial cellulose impregnated with octenidine dihydrochloride-based antiseptic as an antibacterial dressing material for highly-exuding, infected wounds.

The highly absorbent, antibacterial dressings with a sustained release of the antimicrobial are considered necessary measures to counteract chronic wound biofilm-based infections. This study aimed to analyze wet and dry bacterial cellulose (BC) materials, modified by chemical cross-linking, and impregnated with an antiseptic based on octenidine dihydrochloride (OCT) in the context of its antibiofilm/antibacterial activity, exudate absorption, and cytotoxicity. The native BC was obtained from cost-effective, ecological-friendly potato juice (leftover from the starch industry). The ability to absorb and retain OCT, exudate absorption capacity, the kinetics of OCT release as well as antibiofilm/antibacterial activity of modified BC materials against biofilm-forming and planktonic bacteria (Staphylococcus aureus and Pseudomonas aeruginosa) were investigated. The performed analyses revealed that modified BC materials, thanks to their layered structure with numerous air spaces, were characterized by sustained exudate absorption and OCT release profile, which allowed them to exhibit high antimicrobial activity for up to 7 days, with a reduction of planktonic and biofilm cells of 84-100% and 69-93%, respectively. The modified BC materials showed also no cytotoxicity against fibroblast cell line L929 in vitro and were characterized by firm adhesion to the curved surfaces. These results indicate that cross-linked BC impregnated with OCT may be a particularly promising dressing material (obtained using sustainable processes), especially in the treatment of biofilm-infected, highly-exuding wounds.

Chemical Composition and Antibacterial Activity of Liquid and Volatile Phase of Essential Oils against Planktonic and Biofilm-Forming Cells of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen causing life-threatening, hard-to-heal infections associated with the presence of a biofilm. Essential oils (EOs) are promising agents to combat pseudomonal infections because of the alleged antimicrobial activity of their volatile fractions and liquid forms. Therefore, the purpose of this paper was to evaluate the antibacterial efficacy of both volatile and liquid phases of seven EOs (thyme, tea tree, basil, rosemary, eucalyptus, menthol mint, lavender) against P. aeruginosa biofilm and planktonic cells with the use of a broad spectrum of analytical in vitro methods. According to the study results, the antibacterial activity of EOs in their liquid forms varied from that of the volatile fractions. Overall, liquid and volatile forms of rosemary EO and tea tree EO displayed significant antibiofilm effectiveness. The outcomes indicate that these particular EOs possess the potential to be used in the therapy of P. aeruginosa infections.

The effects of rotating magnetic field and antiseptic on in vitro pathogenic biofilm and its milieu.

The application of various magnetic fields for boosting the efficacy of different antimicrobial molecules or in the character of a self-reliant antimicrobial agent is considered a promising approach to eradicating bacterial biofilm-related infections. The purpose of this study was to analyze the phenomenon of increased activity of octenidine dihydrochloride-based antiseptic (OCT) against Staphylococcus aureus and Pseudomonas aeruginosa biofilms in the presence of the rotating magnetic field (RMF) of two frequencies, 5 and 50 Hz, in the in vitro model consisting of stacked agar discs, placed in increasing distance from the source of the antiseptic solution. The biofilm-forming cells' viability and morphology as well as biofilm matrix structure and composition were analyzed. Also, octenidine dihydrochloride permeability through biofilm and porous agar obstacles was determined for the RMF-exposed versus unexposed settings. The exposure to RMF or OCT apart did not lead to biofilm destruction, contrary to the setting in which these two agents were used together. The performed analyses revealed the effect of RMF not only on biofilms (weakening of cell wall/membranes, disturbed morphology of cells, altered biofilm matrix porosity, and composition) but also on its milieu (altered penetrability of octenidine dihydrochloride through biofilm/agar obstacles). Our results suggest that the combination of RMF and OCT can be particularly promising in eradicating biofilms located in such areas as wound pockets, where physical obstacles limit antiseptic activity.

Health benefits and risks associated with element uptake from grilled fish and fish products.

BACKGROUND: The content of essential and toxic elements in grilled fish (Atlantic salmon, trout) and in 20 assortments of fish products was examined. The aim of this study was to assess the fulfilment of the demand for Zn, Fe, Mn and Cu based on recommended dietary allowances (RDAs) or adequate intakes (AIs) and to assess the health risk associated with the consumption of Al, Pb and Cd. The risk assessment was based on estimated weekly intake (EWI), hazard index (HI), target risk ratio (THQ) and percentage: provisional tolerable weekly intake (PTWI) for Al, tolerable weekly intake (TWI) for Cd and reference dose lower bound (BMDL) for Pb. RESULTS: Taking into account the health benefits, the best source of Fe, Cu and Mn was found to be fish products in cans and jars, 150 g of which covered the daily requirement: Fe 9.39%, Cu 2.91% and Mn 1.21%. Smoked fish covered the RDA for Zn to 5.69%. Moreover, the uptake of toxic elements was as follows: Al 0.45% PTWI: Pb 0.74% BMDL10 and Cd 2.20% TWI. The THQ values for Pb and Cd were significantly lower than 1, whereas for Al it reached as high as 4.0. CONCLUSION: The obtained results indicate that there is no risk related to the intake of Pb and Cd with the consumption of the tested fish products. Low consumption of fish by Poles, however, contributes to their small share in covering the demand for microelements, i.e. Zn, Fe, Mn and Cu. © 2021 Society of Chemical Industry.

The Effect of Rotating Magnetic Field on Susceptibility Profile of Methicillin-Resistant Staphylococcus aureus Strains Exposed to Activity of Different Groups of Antibiotics.

Methicillin-resistant strains of Staphylococcus aureus (MRSA) have become a global issue for healthcare systems due to their resistance to most ß-lactam antibiotics, frequently accompanied by resistance to other classes of antibiotics. In this work, we analyzed the impact of combined use of rotating magnetic field (RMF) with various classes of antibiotics (ß-lactams, glycopeptides, macrolides, lincosamides, aminoglycosides, tetracyclines, and fluoroquinolones) against nine S. aureus strains (eight methicillin-resistant and one methicillin-sensitive). The results indicated that the application of RMF combined with antibiotics interfering with cell walls (particularly with the ß-lactam antibiotics) translate into favorable changes in staphylococcal growth inhibition zones or in minimal inhibitory concentration values compared to the control settings, which were unexposed to RMF. As an example, the MIC value of cefoxitin was reduced in all MRSA strains by up to 42 times. Apart from the ß-lactams, the reduced MIC values were also found for erythromycin, clindamycin, and tetracycline (three strains), ciprofloxacin (one strain), gentamicin (six strains), and teicoplanin (seven strains). The results obtained with the use of in vitro biofilm model confirm that the disturbances caused by RMF in the bacterial cell walls increase the effectiveness of the antibiotics towards MRSA. Because the clinical demand for new therapeutic options effective against MRSA is undisputable, the outcomes and conclusions drawn from the present study may be considered an important road into the application of magnetic fields to fight infections caused by methicillin-resistant staphylococci.

Preparation of Komagataeibacter xylinus Inoculum for Bacterial Cellulose Biosynthesis Using Magnetically Assisted External-Loop Airlift Bioreactor.

The aim of this study was to demonstrate the applicability of a novel magnetically assisted external-loop airlift bioreactor (EL-ALB), equipped with rotating magnetic field (RMF) generators for the preparation of Komagataeibacterxylinus inoculum during three-cycle repeated fed-batch cultures, further used for bacterial cellulose (BC) production. The fermentation carried out in the RMF-assisted EL-ALB allowed to obtain an inoculum of more than 200× higher cellular density compared to classical methods of inoculum preparation. The inoculum obtained in the RMF-assisted EL-ALB was characterized by a high and stable metabolic activity during repeated batch fermentation process. The application of the RMF-assisted EL-ALB for K. xylinus inoculum production did not induce the formation of cellulose-deficient mutants. It was also confirmed that the ability of K. xylinus to produce BC was at the same level (7.26 g/L of dry mass), regardless of inoculum age. Additionally, the BC obtained from the inoculum produced in the RMF-assisted EL-ALB was characterized by reproducible water-related properties, mechanical strength, nano-fibrillar structure and total crystallinity index. The lack of any negative impact of inoculum preparation method using RMF-assisted EL-ALB on BC properties is of paramount value for its future applications, including use as a biomaterial in tissue engineering, wound healing, and drug delivery, where especially BC liquid capacity, nanostructure, crystallinity, and mechanical properties play essential roles.

Rotating Magnetic Field Increases ß-Lactam Antibiotic Susceptibility of Methicillin-Resistant Staphylococcus aureus Strains.

Methicillin-resistant strains of Staphylococcus aureus (MRSA) have developed resistance to most ß-lactam antibiotics and have become a global health issue. In this work, we analyzed the impact of a rotating magnetic field (RMF) of well-defined and strictly controlled characteristics coupled with ß-lactam antibiotics against a total of 28 methicillin-resistant and sensitive S. aureus strains. The results indicate that the application of RMF combined with ß-lactam antibiotics correlated with favorable changes in growth inhibition zones or in minimal inhibitory concentrations of the antibiotics compared to controls unexposed to RMF. Fluorescence microscopy indicated a drop in the relative number of cells with intact cell walls after exposure to RMF. These findings were additionally supported by the use of SEM and TEM microscopy, which revealed morphological alterations of RMF-exposed cells manifested by change of shape, drop in cell wall density and cytoplasm condensation. The obtained results indicate that the originally limited impact of ß-lactam antibiotics in MRSA is boosted by the disturbances caused by RMF in the bacterial cell walls. Taking into account the high clinical need for new therapeutic options, effective against MRSA, the data presented in this study have high developmental potential and could serve as a basis for new treatment options for MRSA infections.

The High Impact of Staphylococcus aureus Biofilm Culture Medium on In Vitro Outcomes of Antimicrobial Activity of Wound Antiseptics and Antibiotic.

The staphylococcal biofilm-based infections of wounds still pose a significant therapeutical challenge. Treated improperly, they increase the risk of limb amputation or even death of the patient. The present algorithms of infected wound treatment include, among others, the application of antiseptic substances. In vitro wound biofilm models are applied in order to scrutinize their activity. In the present work, using a spectrum of techniques, we showed how the change of a single variable (medium composition) in the standard in vitro model translates not only to shift in staphylococcal biofilm features but also to the change of efficacy of clinically applied wound antimicrobials such as octenidine, polyhexamethylene biguanide, chlorhexidine, hypochlorite solutions, and locally applied gentamycin. The data presented in this study may be of a pivotal nature, taking into consideration the fact that results of in vitro analyses are frequently used to propagate application of specific antimicrobials in hospitals and ambulatory care units.

The Impact of Intraspecies Variability on Growth Rate and Cellular Metabolic Activity of Bacteria Exposed to Rotating Magnetic Field.

Majority of research on the influence of magnetic fields on microorganisms has been carried out with the use of different species or different groups of microorganisms, but not with the use of different strains belonging to one species. The purpose of the present study was to assess the effect of rotating magnetic fields (RMF) of 5 and 50 Hz on the growth and cellular metabolic activity of eight species of bacteria: Staphylococcus aureus, Pseudomonas aeruginosa, Proteus mirabilis, Klebsiella pneumoniae, Enterococcus faecalis, Enterobacter cloacae, Moraxella catarrhalis, and Bacillus cereus. However, contrary to the research conducted so far, each species was represented by at least four different strains. Moreover, an additional group of S. aureus belonging to a single clonal type but representing different biotypes was also included in the experiment. The results showed a varied influence of RMF on growth dynamics and cellular metabolic activity, diversified to the greatest extent in dependence on the bacterial strain exposed to the RMF and to a lesser extent in dependence on the frequency of the generated magnetic field. It was found that, with regard to the exposed strain of the same species, the effect exerted by the RMF may be positive (i.e., manifests as the increase in the growth rate or/and cellular metabolic activity) or negative (i.e., manifests as a reduction of both aforementioned features) or none. Even when one clonal type of S. aureus was used, the results of RMF exposure also varied (although the degree of differentiation was lower than for strains representing different clones). Therefore, the research has proven that, apart from the previously described factors related primarily to the physical parameters of the magnetic field, one of the key parameters affecting the final result of its influence is the bacterial intraspecies variability.

Potato Juice, a Starch Industry Waste, as a Cost-Effective Medium for the Biosynthesis of Bacterial Cellulose.

In this work, we verified the possibility of valorizing a major waste product of the potato starch industry, potato tuber juice (PJ). We obtained a cost-effective, ecological-friendly microbiological medium that yielded bacterial cellulose (BC) with properties equivalent to those from conventional commercial Hestrin-Schramm medium. The BC yield from the PJ medium (>4 g/L) was comparable, despite the lack of any pre-treatment. Likewise, the macro- and microstructure, physicochemical parameters, and chemical composition showed no significant differences between PJ and control BC. Importantly, the BC obtained from PJ was not cytotoxic against fibroblast cell line L929 in vitro and did not contain any hard-to-remove impurities. The PJ-BC soaked with antiseptic exerted a similar antimicrobial effect against Staphylococcus aureus and Pseudomonas aeruginosa as to BC obtained in the conventional medium and supplemented with antiseptic. These are very important aspects from an application standpoint, particularly in biomedicine. Therefore, we conclude that using PJ for BC biosynthesis is a path toward significant valorization of an environmentally problematic waste product of the starch industry, but also toward a significant drop in BC production costs, enabling wider application of this biopolymer in biomedicine.

Bacterial Nanocellulose Fortified with Antimicrobial and Anti-Inflammatory Natural Products from Chelidonium majus Plant Cell Cultures.

In this work we developed a bi-functional Bacterial-Nano-Cellulose (BNC) carrier system for cell cultures of Chelidonium majus-a medicinal plant producing antimicrobial compounds. The porous BNC was biosynthesized for 3, 5 or 7 days by the non-pathogenic Komagataeibacter xylinus bacteria and used in three forms: (1) Without removal of K. xylinus cells, (2) partially cleaned up from the remaining K. xylinus cells using water washing and (3) fully purified with NaOH leaving no bacterial cells remains. The suspended C. majus cells were inoculated on the BNC pieces in liquid medium and the functionalized BNC was harvested and subjected to scanning electron microscopy observation and analyzed for the content of C. majus metabolites as well as to antimicrobial assays and tested for potential proinflammatory irritating activity in human neutrophils. The highest content and the most complex composition of pharmacologically active substances was found in 3-day-old, unpurified BNC, which was tested for its bioactivity. The assays based on the IL-1ß, IL-8 and TNF-α secretion in an in vitro model showed an anti-inflammatory effect of this particular biomatrix. Moreover, 3-day-old-BNC displayed antimicrobial and antibiofilm activity against Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. The results of the research indicated a possible application of such modified composites, against microbial pathogens, especially in local surface infections, where plant metabolite-enriched BNC may be used as the occlusive dressing.

Superabsorbent crosslinked bacterial cellulose biomaterials for chronic wound dressings.

In this work, we present a novel ex situ modification of bacterial cellulose (BC) polymer, that significantly improves its ability to absorb water after drying. The method involves a single inexpensive and easy-to-perform process of BC crosslinking, using citric acid along with catalysts, such as disodium phosphate, sodium bicarbonate, ammonium bicarbonate or their mixtures. In particular, the mixture of disodium phosphate and sodium bicarbonate was the most promising, yielding significantly greater water capacity (over 5 times higher as compared to the unmodified BC) and slower water release (over 6 times as compared to the unmodified BC). Further, our optimized crosslinked BC had over 1.5x higher water capacity than modern commercial dressings dedicated to highly exuding wounds, while exhibiting no cytotoxic effects against fibroblast cell line L929 in vitro. Therefore, our novel BC biomaterial may find application in super-absorbent dressings, designed for chronic wounds with imbalanced moisture level.

Significant enhancement of citric acid production by Yarrowia lipolytica immobilized in bacterial cellulose-based carrier.

The aim of this study was to improve the yield of citric acid (CA), an industrially valuable metabolite, obtained during Yarrowia lipolytica yeast culturing. To this end, Y. lipolytica cells were immobilized on a novel bacterial cellulose (BC) based carrier and subjected to four subsequent cycles of fed-batch culturing. During the fermentation process, yeasts metabolic stability, glucose consumption and CA production were analyzed. The results of our study have shown that BC-immobilized yeasts utilized more glucose than free cells and that the metabolic activity of BC-immobilized cells and the resultant CA production remained on a stable level throughout 4 fermentation batches, while the drop in free cells' metabolic stability and the consequent drop in CA production was observed with each subsequent batch. Also, the overall concentration of CA product was higher in immobilized vs. free yeasts (121-129 g/L vs. 99-110 g/L, respectively). The presented results indicate that the application of a BC carrier for Y. lipolytica culturing correlates not only with a higher yield of CA product but also with more stable and repeatable conditions of the biotechnological fermentation process. The results obtained in this study may find multiple biotechnological applications in which immobilization of various types of cells is required.