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