Antibacterial 316L Stainless Steel Containing Silver and Niobium

Rare Metal Materials and Engineering Volume 42, Issue 10, October 2013 Online English edition of the Chinese language journal Cite this article as: Rare Metal Materials and Engineering, 2013, 42(10): 2004-2008.

ARTICLE

Antibacterial 316L Stainless Steel Containing Silver and Niobium Yuan Junping1,2, 1

Li Wei1 2

Jinan University, Guangzhou 510632, China; Guangzhou Panyu Polytechnic, Guangzhou 511483, China

Abstract: Ag and Nb were chosen as modifying elements, and their effects on the microstructure, antibacterial property and corrosion resistance of 316L were studied by antibacterial experiment, electrochemical experiment and microscopic analysis. The results show that niobium can refine the grains of 316L effectively and improve the distribution of silver in the matrix. The 316L stainless steel containing an appropriate amount of silver has an excellent antibacterial efficacy to S. aureus and its microbiological corrosion resistance is improved while an excessive silver is prone to form segregation and degrade the corrosion resistance. When 316L the stainless steel contains about 0.04wt%~0.06 wt% Ag and 0.1 wt% Nb, an optimized matching among microstructure, corrosion resistance and antibacterial properties can be obtained. Key words: 316L stainless steel; silver; niobium; microstructure; corrosion resistance; antibacterial performance

Jewelries often carry a large number of bacteria, and so there exists the risk of infection[1]. It would be significantly beneficial if jewelry material itself has antibacterial property. As is known, due to its excellent corrosion resistance, good formability, elegant metallic luster and rich surface colorability, 316L stainless steel has been widely used in jewelry industry and becomes one of the main fashion jewelry materials, especially for piercing jewelry. Since stainless steel itself has no antibacterial ability, it has become a research hotspot whether any antibacterial property, by doping other elements, can be achieved without scarification of its physical properties[2]. Silver was found to own excellent antimicrobial ability and has been extensively used in inorganic antibacterial materials[3]. However, because the solubility of silver in Fe-based alloys under a common condition is very limited[4], the applications of silver in antibacterial stainless steels are mainly through surface modification methods[5]. For example, some researchers implanted silver ions into the surface of stainless steels by an ion-implantation technique and acquired good antibacterial properties[6]. However, such modified surface layer was very thin, once which was worn off, the base material lost the antibacterial ability. By contrast, silver-bearing antimicro-

bial stainless steels have not been studied broadly. Yokota et al[7] studied the effect of continuous casting speed and vanadium on the antimicrobial property of Ag-bearing 430 stainless steel. Liao et al[8] preliminarily studied the effect of Ag on 304 stainless steel. Unfortunately, no in-depth report can be found about Ag-bearing antibacterial 316L stainless steel up till now. Therefore the effects of silver and niobium on the microstructure, the antibacterial property and the corrosion resistance of 316L stainless steel have been investigated in this paper.

1

Experiment

Commercial 316L stainless steel strip, Nb-Fe alloy shots and Ag-Mn alloy shots were chosen as starting materials. Button samples were melt in WK-Ⅱvacuum arc furnace. The practical amount of Ag and Nb for each sample were analyzed with Thermo ARL QUANT' X spectrum analyzer, as shown in Table 1. The button samples were hot forged into sheets of about 2 mm thickness and solid solution treated at 1050 °C for 30 min. Dics (Φ25 mm) or square (5 mm×5 mm) specimens were ground with 1200# sandpapers and cleaned with an ultrasonic cleaner.

Received date: September 2, 2012 Foundation item: the National Natural Science Foundation of China and Guangdong Province (U1034002) Corresponding author: Yuan Junping, Professorate Senior Engineer, Jewelry Institute of Guangzhou Panyu Polytechnic, Guangzhou 511483, P. R. China, Tel/Fax: 0086-20-84739844, E-mail: [email protected] Copyright © 2013, Northwest Institute for Nonferrous Metal Research. Published by Elsevier BV. All rights reserved.

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Yuan Junping et al. / Rare Metal Materials and Engineering, 2013, 42(10): 2004-2008

Table 1 Practical amount of Ag and Nb in modified 316L stainless steel ( wt%) Sample No.

Ag

A0

-

-

A1

0.036

-

A2

Nb

0.097

A3

0.025

0.096

A4

0.043

0.089

A5

0.062

0.093

A6

0.078

0.092

The metallographic structure was examined with an optical microscope. Immediately after antimicrobial test, the samples were cleaned in de-ionized water bath under ultrasonic vibration for 5 min. The surface topographies were observed with a Hitachi S3400N SEM, and the microdomain composition was analyzed with a Bruker QUANTAX200 EDS. The samples were sterilized at 121 °C in an autoclave for 40 min, and then sterilized once again under sterilamp for 30 min. S. aureus was inoculated into 15 mL nutrient broth (which contained peptone 10.0 g/L, NaCl 5.0 g/L and beef extract 5.0 g/L) as test strains and shaken in wave bed for 20 h at 37 °C. The suspension of bacteria was diluted to 1×106 cfu/mL with PBS buffer solution. Inhibition zone test[9] and thin-film adhering quantitative bacteriostasis test[10] were adopted to examine antibacterial properties. In inhibition zone test, 500 μL of the diluted suspension was evenly smeared on agar plate, and sterilized disc samples were closely sticked on the surface of the plate respectively. After cultured at 37 °C for 24 h, the inhibition zone was directly observed. In thin-film adhering test, 50 μL of the diluted suspension was dropped on each sterilized disc sample. The samples were covered with sterile thin film to spread the bacteria uniformly and kept in a test chamber for 24 h, with a constant temperature of 37 °C and relative humidity over 90%. Each sample was then washed thoroughly with PBS buffer solution, and the eluent was made serial dilution. Agar plate method was used to culture and count live bacteria. All experiments were carried out in triplicate. The relative sterilizing rate of the bacteria was determined according to formula (1): R=(C–A)/C×100% (1) where R is the relative sterilizing rate, C is the mean number of bacteria on the control sample, and A is the mean number of bacteria on the analyzed sample. The potentiodynamic polarization studies were carried out with LK2005 electrochemical workstation. Three-electroded system was adopted, the test surface was used as the working electrode, the saturated calomel electrode (SCE) as the reference electrode, and the platinum foil as the counter electrode. The electrolyte was artificial sweat, which consisted of 1.00 g/L of urea, 5.00 g/L of sodium chloride, and 940 mL/L of lactic acid, and the pH value was 6.50. After immersed in arti-

ficial sweat for 10 min to attain a stable open circuit potential, the samples were examined in the potential range of –1000 to 1000 mV and at a scan rate of 5 mV/s. To study the effect of Ag and microbial environment on corrosion resistance, two groups of samples were prepared. Group 1 was in an original state, while group 2 was soaked in 1×106 cfu/ml suspension of S. aureus and kept at 37 °C for 24 h, ultrasonic cleaned in de-ionized water bath for 5 min; finally their polarization curves were examined in the same way.

2

Results and Discussion

2.1 Metallographic structure and distribution of Ag Fig.1 shows the metallographic structure of sample A0 and A2. It can be seen when trace of niobium is added to 316L stainless steel, the microstructure of A2 is obviously finer than that of commercial 316L (A0). As is known, Nb is an element forming an extremely strong carbide or nitride, which can not only help to hold back the intergranual corrosion of stainless steel, but is an effective grain refiner during solidification and recrystallization. Particularly in the process of controlled rolling and normalizing, Nb can strongly inhibit austenite recrystallization and markedly refine the grain[11]. Fig.2 shows the distribution of Ag in different samples. There are some clusters of silver in A1, and the segregation of silver in A5 is also obvious. By comparison, the distribution of silver in A4 is uniform on the whole. As is known, the solid solution of silver in α-Fe is extremely low[4]. Although 316L stainless steel is austenitic, the b

a

100 μm Fig.1 Microstructure of sample A0 (a) and A2 (b) a

b

c

20 μm Fig.2 Distribution of Ag in sample A1 (a), A4 (b) and A5 (c)

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solubility of Ag is still very limited, and therefore, Ag is prone to form segregation when the addition of Ag is greater than its solubility, especially when the grain is coarse. In sample A1, 0.036wt% Ag is added solely, its grain is relatively coarser, which is unfavorable for uniform distribution of silver. When trace of silver and niobium are added together, the distribution of silver changes. There is no visible segregation of silver in A4 although its silver content (0.043 wt% Ag) is more than A1. However, when silver content increases to a certain amount, the segregation of silver appears again. It can be inferred that the grain refining role of niobium is beneficial for alleviating segregation of silver. Silver can be distributed uniformly in the matrix when its content is low, but if it exceeds a certain amount, the segregation of silver can not be completely restrained by the role of niobium. 2.2 Effect of Ag on antimicrobial property Fig.3 shows the inhibition zone test results. As can be seen, the inhibition zone of sample A0 to S. aureus on ordinary nutrition agar plate is zero, and no bacteriostasis is shown. While for sample A5, the inhibition zone is just only recognizable and no obvious antimicrobial efficacy is shown either. Fig.4 shows the growth of S. aureus in agar plate surface after thin-film adhering test. By counting the bacterial colony number, the average sterilization rate can be calculated, as shown in Table 2. As can be seen, when silver content is only 0.025 wt% (A3), the modified 316L stainless steel shows some moderate antimicrobial efficacy. With the increasing of silver content, the antibacterial performance is continuously improved. When silver content reaches 0.043 wt% (A4) and 0.062 wt% (A5), the sterilization rates of both samples are more than 99%, showing excellent antibacterial performance to S. aureus. However, further increasing of silver content would slightly weaken antimicrobial efficacy (see A6 in Table 2). Fig.5 shows the surface topographies of A0 and A5 after antimicrobial test. There is a thin gray film with many obvious corroded pits on sample A0 surface, and sediments can be hardly seen. As is known, when common stainless steel is soaked in suspension of bacteria, bacteria will adhere on the surface and form a biological film. The metabolization activity of bacteria can change the microenvironment of the interface between the biological film and the steel surface, and then, a

a

b

c

d

e

Fig.4 Antibacterial effect of each sample: (a) A0, (b) A3, (c) A4, (d) A5, and (e) A6 Table 2

Average sterilization rate to S. aureus

Sample No.

Average sterilization rate/%

A0 A3 A4 A5 A6

67.4 99.3 100 98.5 a

b

b

Fig.5 Surface topographies after antibacterial test: (a) A0 and (b) A5

Fig.3 Inhibition zones of sample A0 (a) and A5 (b)

both the corrosion potential and the stability of the surface passive film are affected, leading to microbial corrosion[12,13]. Sample A0 has no antibacterial efficacy; therefore, the metabolism of bacteria could not be restrained, leading to obvious corroded pits on the surface. When sample A5 contacts

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the suspension of S. aureus for a period of time, its surface topography is significantly different from that of A0; there appear large amounts of sediments on the surface, and corroded pits are much less. To fully understand these sediments, a randomly selected sediment on sample A5 was observed at a high magnification, as shown in Fig.6. The composition is shown in Table 3. It can be seen that the sediments contain a large amount of oxygen and phosphorus, and traces of nitrogen, calcium, sulfur, etc. Since these detected elements are the basic components of biological tissues in bacteria, it can be inferred that the sediments should be the killed bacteria debris.

5 μm Fig.6 Sediments in A5

Table 3 Main composition at the area covered by sediments in Fig.6 (wt%) Element

Fe

Cr

Ni

Mo

Mn

C

N

O

P

S

Si

Ca

Nb

Ag

Content

51.56

13.08

9.01

1.59

0.47

0.02

0.06

22.78

0.71

0.07

0.55

0.03

0.03

0.04

4 2 log(I/mA)

0 –2

A32 A42 A41

A61 A51 A31

–4 –6

A52

A62 A02

A01

–8 –0.9

–0.6

–0.3

0.0

0.3

E/V Fig.7 Polarization curves in artificial sweat 0.1 0 Potentials/V

Contrasting the test results of two antibacterial experiments in this study, it is seen that sample A5 does not show rudimentary antibacterial property in the inhibition zone test, while it displays excellent antibacterial efficacy in thin-film adhering test. It indicates that only through direct contacting with bacteria can Ag-bearing stainless steel really realize antibacterial efficacy. Therefore, it is inferred the antibacterial mechanism of Ag-bearing stainless steel is the synthesized effects of electrostatic adhesion of silver ions and their reacting with bacteria. When Ag-bearing stainless steel contacts the suspension of bacteria, it will release partial silver ions. Since the cell wall and membrane of S. aureus are negatively charged, the silver ions can be adhered to the cell wall in consequence of the electrostatic attraction[14], which will constrain the activity and disorder the survival microenvironment of bacteria. What is more, silver ions can penetrate through the wall and enter the cell, which will react with functional groups of protein, such as sulfydryl or amino, and destroy the enzyme system of the cell membrane[15], making bacteria lose proliferation capacity and die eventually. 2.3 Effect of Ag on corrosion resistance of 316L Fig.7 shows the polarization curves of the samples in artificial sweat, A01~A41 represent the results before soaking in suspension of bacteria, and A02~A42 represent those after soaking. Fig. 8 shows the self corrosion potentials and pitting potentials, SCP1 and PP1 represent self corrosion potential and pitting potential, respectively before soaking, while SCP2 and PP2 represent after soaking. As can be seen from Fig.8 that the self corrosion potential and pitting potential of sample A3 and A4 are slightly higher than that of A0 before soaking in suspension of S. aureus, and A5 is quite close to A0, while both the potentials of A6 decrease to a certain degree. It is suggested that low silver content could slightly improve corrosion resistance of 316L stainless steel in non microbial environment. When silver increases to a certain amount, it brought an adverse effect.

–0.1

SCP1

PP1

–0.2

SCP2

PP2

–0.3 –0.4 –0.5 –0.6 0

0.02

0.04

0.06

0.08

Ag/wt% Fig.8 Effect of Ag on corrosion potentials

After soaking in suspension of S. aureus for 24 h, both self corrosion potential and pitting potential increase first and then drop down with the increase of silver content, and the corresponding Ag contents to the peaks are 0.043wt% and 0.062wt%, respectively. By contrast, the potentials of sample A0 obviously descend after soaking, and the reduction extent is the biggest among all samples. When 316L contains 0.025

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wt% Ag (sample A3), the reduction rate of self corrosion potential diminishes, while its pitting potential is quite close to that before soaking. After Ag content reaches 0.043 wt% (sample A4), the self corrosion potential is only slightly lower than the original value, instead, pitting potential is higher, especially for sample A5. It is conjectured that the adhered bacteria are killed partially or entirely upon contacting Agbearing 316L stainless steel, and the bacteria debris form sedimentary layer, which could hinder further microbiological corrosion. After ultrasonic cleaning, living bacteria are desorbed, while those solid sedimentary films continue to wrap the sample surface as a barrier. Since samples A4, A5 and A6 have excellent antimicrobial efficacy, there are a lot of sediments on their surfaces; therefore, their corrosion potentials show a significant increase in the electrochemical test.

3

Conclusions

1) By the aid of the grain refinement effect of niobium, a trace of silver can be evenly distributed in the matrix of 316L and slightly improve its corrosion resistance in non microbial environment, but when silver content is more than a certain amount, segregation is prone to form and corrosion resistance would degrade. 2) 316L stainless steel would suffer obvious corrosion in 1×106 cfu/mL suspension of S. aureus for 24 h at 37 °C. An appropriate amount of silver added to the stainless steel could obtain excellent sterilization effect to S. aureus and improve its microbiological corrosion resistance. 3) The antibacterial efficacy of silver-bearing stainless steel can be realized through direct contacting with bacteria. 4) When 316L stainless steel contain about 0.04wt%~ 0.06wt% Ag and 0.1 wt% Nb, an optimized matching among microstructure, corrosion resistance and antibacterial properties can be obtained.

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