Production and applications of microbially precipitated nanosilver

Microbial contaminations worldwide give rise to problems in many areas going from water treatment to medical applications and food preparation. Hence, there is an increasing demand for biocides to control such contaminations and their concomitant economical and health impact. Since Antiquity, silver has been used for its antimicrobial properties. However, in the twentieth century silver was set aside by antibiotics. Currently microbial resistance towards current antibiotics increases and toxic disinfection byproducts produced by conventional decontamination methods have been observed. As a result, the interest to use silver as disinfectant has re-emerged. Silver has the advantage of having a broad antimicrobial spectrum against Gram-positive and Gram-negative bacteria, fungi and viruses, and it has a relatively low toxicity towards humans. Recently, silver is applied more and more in the form of nanoparticles because of the high specific surface of such particles which results in an increased activity. For the synthesis of nanosilver, several chemical and physical methods exist. These methods, however, have disadvantages such as high production costs, low scalability and a broad size distribution of the produced nanoparticles. Also the instability of the nanoparticles upon application can be problematic since aggregation lowers the antimicrobial activity by decreasing the specific surface. Moreover, there is a need for “green” production processes which omit the use of solvents and toxic reagents. Hence, the biological production of nanosilver is of interest. Lactobacillus sp. are GRAS bacteria, which are known to produce exopolysaccharides rich of reducing sugars. In the past, these bacteria were used to remove heavy metals from (waste)water by biosorption. Previously it was also observed that they could reduce Ag+ to Ag0. In Chapter 2, it was investigated if Ag+ reduction also resulted in nanoparticle formation and if reduction could occur in alkaline conditions in the presence of high silver concentrations (1g/L). Ag+ reduction by Lactobacillus sp. was compared with that of other lactic acid bacteria (LAB), and that of few other Gram-positive and Gram-negative bacteria which do not belong to group of LAB. Lactobacillus sp. and also the other LAB were all able to reduce Ag+ to its metallic form, while the other bacteria could not. The pH was of importance for the amount of silver recovered after reduction and for the rate of the reaction. At pH 11.5, L. fermentum reduced Ag+ within one minute and recovered 83% of the initially added silver concentration. Transmission electron microscopy revealed that L. fermentum also produced the smallest nanoparticles (11.2 nm) with the most narrow size distribution compared to four other Lactobacillus strains. Moreover, the nanoparticles were mainly distributed on the cell wall. This makes biogenic silver produced by L. fermentum the most interesting for antimicrobial applications. In a second part, L. fermentum was further used for production of biogenic silver at larger scale. The bacteria were grown in different reactors ( 1L-Erlenmeyer vs. 5L-fermentor) and different types of growth medium (MRS vs. LFM) and differences in biomass production and biogenic silver yield were investigated. When grown in Erlenmeyers, the silver recovery or yield was the highest. However, these reactors delivered much less biomass and for largescale production they are unpractical. Hence, cultivating L. fermentum in a fermentor is more realistic. In this case, the highest silver recovery (55%) was obtained when the bacteria were grown in LFM. When the biomass production would be optimized to 16 g/L CDW, the production cost of biogenic silver would be in the order of 4419 €/kg. Since the antimicrobial activity is related to the size of the nanoparticles, the nanosilver or biogenic silver produced by L. fermentum seemed the most interesting for antimicrobial applications. This was confirmed by the high antibacterial activity and antiviral activity, as observed in Chapter 4 and Chaper 5. In addition to the nanoparticle size, the presence of the bacterial carrier was of great importance for the antimicrobial activity. The bacterial cell on which the nanoparticles are precipitated served as a scaffold which stabilized the nanoparticles and prevented them from aggregations. This resulted in a maintenance of the high specific surface, hence a high antimicrobial activity. This was in contrast with chemically produced nanoparticles which clustered upon addition to drinking water or liquid broth; they only could inhibit bacteria when much higher concentration were used. The main mode of action of biogenic silver was the release of silver ions. The stabilization of the nanoparticles by the bacterial carrier stimulated the release of ions, while in the case of chemically produced nanosilver, the decreased specific surface resulted in a diminished release of ions. Although reactive oxygen species production was observed, it did not seem to contribute to the antibacterial activity, nor did direct physical contact between the biogenic nanoparticles and the bacteria. This resulted in an antibacterial activity of biogenic silver which was comparable with or a bit lower than ionic silver. In contrast, the antivral activity was higher for biogenic silver than for ionic silver. Biogenic silver damaged the viral capsid resulting in an decreased infectivity of the viruses. The exact mechanism, however, is unknown and needs to be examined further. In Chapter 6, it was examined if a bacterial carrier also could serve as a Trojan horse to trick Acanthamoeba castellanii to feed on bacteria loaded with silver nanoparticles. This resulted in an increased inhibition of the amoebae compared to ionic silver. However, when the silver concentration is below the inhibitory concentration (100 mg/L), the amoebae still could use the bacterial carrier as energy source and reproduce themselves. Therefore, further research is needed to improve the uptake of silver nanoparticles functionalized with bacterial structures, without stimulating amoebal reproduction. To conclude, the production of biogenic silver using bacteria is not only beneficial for the environment, but also results in creating nanoparticles with an added value compared to chemically produced silver nanoparticles. The application field of biogenic silver, however, warrants further exploration. Hence, future research needs to point out in which areas biogenic silver can be effectively applied as antimicrobial, but also as catalyst

Enhanced disinfection efficiencies of solar irradiation by biogenic silver

Solar disinfection (SODIS) is a very inexpensive and easy-to-run water-treatment process that is widely used in third world countries to decrease the number of waterborne diseases and mortality. However, it does have a number of disadvantages, including the long time needed for complete disinfection, especially during cloudy days, and the possible regrowth of germs during subsequent storage of the water. We tested whether the addition of low concentrations of biogenic silver, which is nanosilver produced on a bacterial scaffold of Lactobacillus fermentum, to the treatment process would improve the disinfection process in general and, more specifically, retard the growth of germs during water storage. Biogenic silver was found to accelerate the inactivation of Escherichia coli by SODIS by approximately twofold. This effect was more pronounced during the first 3 h of the disinfection process and was better than when TiO2 was added. Biogenic silver which was immobilized on zeolite or polysulphone (PSF) to create a reusable formulation enhanced SODIS during the first 3 h, with the Ag-PSF formulation giving the best results. Ag-PSF released silver more slowly to the surrounding water, making it a more suitable formulation for drinking water disinfection, and it prevented germ regrowth during storage of the treated water

The antibacterial activity of biogenic silver and its mode of action

In a previous study, biogenic silver nanoparticles were produced by Lactobacillus fermentum which served as a matrix preventing aggregation. In this study the antibacterial activity of this biogenic silver was compared to ionic silver and chemically produced nanosilver. The minimal inhibitory concentration (MIC) was tested on Gram-positive and Gram-negative bacteria and was comparable for biogenic silver and ionic silver ranging from 12.5 to 50 mg/L. In contrast, chemically produced nanosilver had a much higher MIC of at least 500 mg/L, due to aggregation upon application. The minimal bactericidal concentration (MBC) in drinking water varied from 0.1 to 0.5 mg/L for biogenic silver and ionic silver, but for chemically produced nanosilver concentrations, up to 12.5 mg/L was needed. The presence of salts and organic matter decreased the antimicrobial activity of all types of silver resulting in a higher MBC and a slower inactivation of the bacteria. The mode of action of biogenic silver was mainly attributed to the release of silver ions due to the high concentration of free silver ions measured and the resemblance in performance between biogenic silver and ionic silver. Radical formation by biogenic silver and direct contact were found to contribute little to the antibacterial activity. In conclusion, biogenic nanosilver exhibited equal antimicrobial activity compared to ionic silver and can be a valuable alternative for chemically produced nanosilver

Biogenic silver for disinfection of water contaminated with viruses

The presence of enteric viruses in drinking water is a potential health risk. Growing interest has arisen in nanometals for water disinfection, in particular the use of silver-based nanotechnology. In this study, Lactobacillus fermentum served as a reducing agent and bacterial carrier matrix for zerovalent silver nanoparticles, referred to as biogenic Ag-0. The antiviral action of biogenic Ag-0 was examined in water spiked with an Enterobacter aerogenes-infecting bacteriophage (UZ1). Addition of 5.4 mg liter(-1) biogenic Ag-0 caused a 4.0-log decrease of the phage after 1 h, whereas the use of chemically produced silver nanoparticles (nAg(0)) showed no inactivation within the same time frame. A control experiment with 5.4 mg liter(-1) ionic Ag+ resulted in a similar inactivation after 5 h only. The antiviral properties of biogenic Ag-0 were also demonstrated on the murine norovirus 1 (MNV-1), a model organism for human noroviruses. Biogenic Ag-0 was applied to an electropositive cartridge filter (NanoCeram) to evaluate its capacity for continuous disinfection. Addition of 31.25 mg biogenic Ag-0 m(-2) on the filter (135 mg biogenic Ag-0 kg(-1) filter medium) caused a 3.8-log decline of the virus. In contrast, only a 1.5-log decrease could be obtained with the original filter. This is the first report to demonstrate the antiviral efficacy of extracellular biogenic Ag-0 and its promising opportunities for continuous water disinfection

Thiazolide Prodrug Esters and Derived Peptides: Synthesis and Activity

Amino acid ester prodrugs of the thiazolides, introduced to improve the pharmacokinetic parameters of the parent drugs, proved to be stable as their salts but were unstable at pH > 5. Although some of the instability was due to simple hydrolysis, we have found that the main end products of the degradation were peptides formed by rearrangement. These peptides were stable solids: they maintained significant antiviral activity, and in general, they showed improved pharmacokinetics (better solubility and reduced clearance) compared to the parent thiazolides. We describe the preparation and evaluation of these peptides

Lactic acid bacteria as reducing and capping agent for the fast and efficient production of silver nanoparticles

There is a growing demand for silver-based biocides, including both ionic silver forms and metallic nanosilver. The use of metallic nanosilver, typically chemically produced, faces challenges including particle agglomeration, high costs, and upscaling difficulties . Additionally, there exists a need for the development of a more eco-friendly production of nanosilver. In this study, Gram-positive and Gram-negative bacteria were utilized in the non-enzymatic production of silver nanoparticles via the interaction of silver ions and organic compounds present on the bacterial cell. Only lactic acid bacteria, Lactobacillus spp., Pediococcus pentosaceus, Enterococcus faecium, and Lactococcus garvieae, were able to reduce silver. The nanoparticles of the five best producing Lactobacillus spp. were examined more into detail with transmission electron microscopy. Particle localization inside the cell, the mean particle size, and size distribution were species dependent, with Lactobacillus fermentum having the smallest mean particle size of 11.2 nm, the most narrow size distribution, and most nanoparticles associated with the outside of the cells. Furthermore, influence of pH on the reduction process was investigated. With increasing pH, silver recovery increased as well as the reduction rate as indicated by UV-VIS analyses. This study demonstrated that Lactobacillus spp. can be used for a rapid and efficient production of silver nanoparticles