Influence of the silver nanoparticles on microbial community in different environments *

The aim of this study was to assess the influence of silver nanoparticles (AgNPs) on the number and diversity of microbial community in different environments (soil extract, water, sewage), and to determine whether the environment inhibits or accelerates the influence of AgNPs on moulds. AgNPs (45 ppm) present in the environment decreased bacterial (91%) and fungal (33–85%) numbers, and eliminated some strains, e.g., Alternaria alternata and Cryptococcus laurentii. Based on the biomass growth of Aspergillus niger and Penicillium chrysogenum in a medium with AgNPs and the environmental samples, it was noticed that environment can enhance (soil extract) or inhibit (sewage) antifungal activity of AgNPs.

A large number of products and waste contains silver nanoparticles.AgNPs accumulate in the environment and their wide antimicrobial effects may potentially cause ecological problems; for example, they can cause damage to the soil by eliminating particular species of microorganisms, or interfering with the phosphorous, sulphur and nitrogen cycles (Ratte, 1999).To date, published studies have solely focused on the influence of AgNPs on the total number of microorganisms in soil and wastewater (Schlich et al., 2013;Patil, 2014).However, there are no detailed studies investigating the influence of the environment (water, soil, sewage) on the microbial activity of AgNPs.The aim of this study was to assess the influence of silver nanoparticles on the number and diversity of microbial communities in different environments (soil extract, water, sewage), and determine whether the environment has a protective effect on microorganisms (particularly moulds) or enhances the antimicrobial effect of AgNPs.

MATERIALS AND METHODS
Environmental samples.Water, soil extract and sewage were used for environmental sample testing (Section "Influence of AgNPs on the number and diversity of the microbial community in water, soil extract and sewage") and studies using model organisms (Section "Influence of the environment (water, soil extract, sewage) on the antifungal properties of AgNPs").Water was sampled from floodplain ponds of the Sokołówka river in Łódź,Poland (51°48'16.9"N;19°26'39.3"E).Soil was sampled from farmland (rye) in Syberia,Poland (51°50'26.5"N;19°45'45.7"E).Sewage was sampled from a grit chamber of the Group Wastewater Treatment Plant, Łódź,Poland (51°43'40.6"N;19°20'51.6"E).Water and sewage were filtered on cellulose filters (Filtrak, Germany) to separate impurities.Extracts from soil samples were prepared by suspending 10 g of soil in 100 ml of sterile distilled water, shaking (30 min, 200 rpm) and filtering on cellulose filters (Filtrak, Germany).
Microorganisms.We used fungi as the study model.Stocks were obtained from the American Type Culture Collection (ATCC) and Pure Culture Collection at the Institute of Fermentation Technology and Microbiology, Lodz University of Technology (Łódzki Ośrodek Czystych Kultur -ŁOCK).The following organisms were used: Aspergillus niger ATCC 16404 (Minimum In-hibitory Concentration (MIC) 22.5 ppm) and Penicillium chrysogenum ŁOCK 0531 (MIC 45 ppm).Minimum inhibitory concentrations (MIC) for fungi were established in previous studies (Gutarowska et al., 2012).We selected the above strains as they are frequently isolated from the tested environments, and the lack of research regarding the influence of AgNPs on moulds in these environments.
Influence of AgNPs on the number and diversity of the microbial community in water, soil extract and sewage.The effect of silver nanoparticles on the number of microorganisms in water, soil extract and sewage was determined by adding AgNPs (45 ppm -established in previous studies) to the environmental samples and incubating for 24 h at 30±2°C.Microbial numbers in all samples (with and without silver nanoparticles) were determined using serial dilutions in sterile saline (0.85% NaCl), and culturing on microbiological media: MEA (Malt Extract Agar, Merck, Germany) with chloramphenicol for fungi, and TSA (Tryptic Soy Agar, Merck Germany) with nystatin for bacteria.Samples were incubated using the following conditions: bacteria 24 h, 30±2°C, and fungi 72 h, 27±2°C.After incubation, the microbial colonies were counted and expressed as cfu/ml.All samples were done in triplicate.
The reduction in microorganism number (total and particular species) due to AgNPs treatment was calculated using equation (1): where: N is the number of microorganisms in the sample with silver nanoparticles (cfu/100 ml); N 0 is the number of microorganisms in the sample without silver nanoparticles (cfu/100 ml).
A one-way ANOVA was performed to assess statistical significance between the number of microorganisms (cfu/100 ml) before and after AgNPs treatment.
Influence of the environment (water, soil extract, sewage) on the antifungal properties of AgNPs.To determine the influence of AgNPs on the growth of the moulds: A. niger ATCC 16404 and P. chrysogenum ŁOCK 0531, the following media were used: 1) MEB (Malt Extract Broth, Merck, Germany); 2) MEB with AgNPs (22.5-45 ppm depending on the strain); 3) MEB with environmental samples (water, soil extract, sewage); 4) MEB with environmental samples and AgNPs.
MEB and the particular environmental sample were mixed in a ratio of 1:1.
Flasks containing 50 ml of the appropriate medium were inoculated (100 μl) with moulds (inoculum concentration: 10 6 cfu/ml).Samples were incubated for 14 days at 27±2°C.The environmental impact on mould growth was determined by measuring the biomass of the mould after 3, 7 and 14 days.The samples were filtered on cellulose filters (Filtrak, Germany), and the dry matter content was quantified using the gravimetric method (MAC 110/NH, Radwag, Poland).All samples were done in triplicate.
The reduction in mould biomass, due to AgNPs treatment, was calculated using equation ( 1), where N is the mould biomass.A one-way ANOVA was performed to assess statistical significance between the mould biomass samples (g dry weight) before and after AgNPs treatment.
The addition of silver nanoparticles to environmental samples (soil extract, water and sewage) at a concentration of 45 ppm, showed that bacteria are more susceptible to AgNPs than fungi.AgNPs decreased the microorganisms' number by approximately 1 log unit in a statistically significant manner.The reduction of bacterial numbers (R=90.1-90.8%)was similar in all environments tested.The highest reduction in fungal numbers (R=85.0%)was observed with the sewage sample, average ± standard deviation; R, reduction calculated according to eqn.(1); *statistically significant differences between control samples (without AgNPs) and samples with AgNPs; one-way ANOVA p<0.05; others -unidentified strains with the frequency of occurrence < 20%; *statistically significant differences between control samples (without AgNPs) and samples with AgNPs; one-way ANOVA p<0.05 ×100% R= while the lowest was with the soil extract (R=33.2%)(Table 1).Schlich et al. (2013) tested the influence of silver nanoparticles (NM-300K; < 20 nm) on soil microorganisms and obtained a decrease of 39.9-73.2% in microbial biomass after 7 days.Patil (2014) obtained a higher reduction of 92.9-94.6% in bacterial numbers in sewage following AgNPs application, when compared to us; however, they did not conduct studies on fungi, and used silver nanoparticles biosynthesized by Fusarium semitectum.
The addition of silver nanoparticles to environmental samples eliminated 4 fungal species: Alternaria alternata, Penicillium glabrum, Candida sp., Cryptococcus laurentii.Cellulomonas sp. was the most sensitive species to silver nanoparticles, with a statistically significant reduction above 90% (R=93.2-99.7%),with all environmental samples tested.Some species (A.niger and P. chrysogenum), exhibited different sensitivities to AgNPs, depending on the environment.Mucor hiemalis, Rhodotorula mucilaginosa, Aeromonas hydrophila and Streptomyces sp. were insensitive to AgNPs (Fig. 1).Biocide sensitivity is dependent on the microorganism's genus, species, strain, and also on isolates, which might be due to previous exposure to a biocide containing silver (Gutarowska et al., 2012).Based  on our findings, we hypothesize that the sensitivity of various microorganisms to AgNPs may be linked to environmental properties (protective or enhancing the effectiveness of AgNPs).Therefore, we conducted studies using model microorganisms to determine the influence of AgNPs on mould biomass growth in media, with or without the environmental background.These studies showed that the addition of silver nanoparticles (at MIC) to MEB medium caused a statistically significant reduction in the mycelium biomass of A. niger and P. chrysogenum, starting on the 3 rd day and persisting until the end of the incubation period (14 days), when compared to control samples without AgNPs (Table 2).The decrease in mould biomass was 28-35% (14th day).
The supplementation of MEB with soil extract, water and sewage delayed mould growth, and decreased mycelium biomass by 44-63%, when compared to control samples containing only the MEB medium.MEB with AgNPs containing soil extracts showed the highest reduction (R=35-64%).Similar results were obtained for control (MEB) and MEB with water (R=28-42%).MEB containing sewage with AgNPs showed the lowest biomass reduction (R=13-36%).Results suggest that sewage may inhibit the antimicrobial properties of AgNPs, while soil extract may facilitate them.In future research, environmental components must be analysed focusing on the enhancement and inhibitory properties of AgNPs activity against microorganisms.

Figure 1 .
Figure 1.Microbial diversity of environments before and after AgNPs application