188bet asian Testing antibacterial effect of silver nanoparticles on sulfate-reducing bacteria

Summary

In 188bet asian oil and gas industry, sulfate-reducing bacteria can generate hydrogen sulfide (H2S) in their growth, thus decreasing 188bet asian commercial value of crude oil, causing metal equipment corrosion and seriously affecting 188bet asian economic efficiency and 188bet asian health of workers on drilling rigs. Because of their strong antibacterial and environmental friendly characteristics, silver nanoparticles have been studied and applied in various sectors world-wide. This paper presents some initial research results on 188bet asian synthesis of silver nanoparticles (NPs) with 188bet asian mean silver nanoparticle size of 18 - 21nm being effective to reduce bacterial numbers from 106tb/ml to < 10tb/ml (99%). This activity was tested on sulfate-reducing bacteria EPC-KK2 - Desulfomicrobium baculatum which was isolated from crude oil samples taken from Bach Ho field.

Key words: Silver nanoparticle, biocide, sulfate-reducing bacteria, Bach Ho field.

1.    Introduction

188bet asian sulfate reducing bacteria (SRB) are anaerobic bacteria that produce H2S. This type of bacteria can survive in extreme environments such as high temperature, high pressure, high salinity, to alkaline or acidic environment and is especially quite common in oil fields and exploitation wells. 188bet asian SRB produce H2S that is acidifying, leading to a reduction in 188bet asian commercial value of crude oil, corrosion of metalworking equipment, piping, and 188bet asian health of workers on drilling rigs [1, 2]. Under favourable conditions, desulfurising bacteria can develop biofilm membranes that occlude 188bet asian reservoir, which reduces 188bet asian ability of 188bet asian water to pump.

Currently, popular biocides being used are aldehydes or cyclic amines in combination with cationic active substances, which are highly toxic to humans and 188bet asian environment. When these biocides are used for a long time 188bet asian phenomenon of“grease” will occur. It is therefore in need of a new biocide with better antibacterial effect and being more environmentally friendly.

Silver NPs is a new material that is known for its superior disinfection ability and has many applications in life. Silver NPs are very small in size and have very large surface area. They can penetrate easily into microbial cells, alter biochemical mechanisms, inactivate microbial metabolism, and finally destroy them [3].

188bet asian antibacterial mechanism of silver NPs is 188bet asian  result of 188bet asian conversion of silver atoms on 188bet asian surface of silver NPs to free Ag+ and free ions that will affect bacteria. Some mechanisms of silver NPs action on bacteria are being understood, for example silver ions inhibit 188bet asian ability of oxygen to travel through 188bet asian cell wall due to its ability to bind to 188bet asian peptidoglycan (cell-wall component), leading to cell death [4]. In animals without cell walls, they are unaffected by exposure to these ions. In 188bet asian other mechanism, silver ions can pass through 188bet asian cell membrane into 188bet asian cell and react with sulfhydryl - SH group of 188bet asian oxygen-converting enzyme molecule, neutralising 188bet asian enzyme that inhibits bacterial cell respiration. In addition, silver ions are capable of binding to DNA bases and neutralising 188bet asian charge of phosphate, preventing DNA replication [5 - 8].

Antibacteria with silver NPs is a new research direction in 188bet asian manufacture of biocides for use in 188bet asian oil and gas industry. This article presents some  results  of  synthesis of silver NPs and testing bactericidal ability of silver NPs on desulfurising bacteria isolated from Bach Ho crude oil samples.

2.    Materials and methods

    Materials and equipment

    Materials

In this study, all reagents of producing silver NPs colloids were of analytical grades and used as received without further purification. Silver nitrate AgNO3 (99.98%) was used as 188bet asian silver precursor, which was obtained from  Merck (Germany). Sodium borohydride NaBH4 (96.5%) was obtained from Chemical Ltd., whereas 188bet asian sodium citrate Na3C6H5O7 (99.0%) from Sigma Aldrich was used as producing agents. All 188bet asian aqueous solutions were prepared in double distilled water.

188bet asian improved Postgate’s B bacterial culture medium was used for bacterial culture KH2PO4 0.5g/l; Na2SO4 1g/l; NH4Cl 1g/l; MgSO4 2g/l; NaCl 4g/l; CaCl2 0.1g/l; marine water 200ml; distilled water 800ml; C3H5NaO3 2g/l; pH 7.2g/l; C2H3NaO2.3H2O 1.75g/l;

FeSO4 0.5g/l; yeast extract 0.5g/l; 188bet asian medium should be autoclaved for 15 minutes at 121oC (250oF). 188bet asian pH should be adjusted with NaHCO3.

SRB strain was isolated from MSP10 crude oil sample of Bach Ho field and identified by analysing and sequencing 16S rRNA genes.

    Equipments

188bet asian UV-visible spectra were recorded over 188bet asian range of 200 - 800nm by Thermo Evolution 300. 188bet asian size and morphology of silver nanoparticle colloids were investigated using transmitted electron microscope (TEM) Philips CM 120. Particle size measuring device by laser light scattering LA-950.

    Methods

188bet asian chemical reduction method is used  for  188bet asian production of silver NPs from AgNO3, with 188bet asian reducing agent NaBH4 or Na3C6H5O7 [9]. 188bet asian reduction process for silver NPs is shown in Figure 1.

 Evaluating 188bet asian dispersibility of silver NPs: Silver NPs were diluted at different concentrations and UV-VIS spectra were measured to evaluate 188bet asian existence and contraction of silver NPs.

Isolation and identification of SRB: Bacteria were isolated from Bach Ho oil samples. SRB was isolated on Postgate’s B medium and purified to 188bet asian mono-assay by means of critical dilution. SRB strain was Gram stained and its cell shape was observed under 188bet asian optical microscope.

188bet asian bacterial strain was cultured and enriched, and identified by analysing and sequencing of 16S rRNA genes.

188bet asian biocide efficiency was tested according to bacterial growth after exposure to silver NPs according to API RP38 standard.

3.    Results and discussion

    Preparation of silver nanoparticle solution

    Selection of reducing agents

188bet asian results in Figures 2 and 3 show that 188bet asian particle sizes of products obtained from Na3Cit (Na3C6H5O7) reductant are larger than those from NaBH4. When Na3Cit was used, 188bet asian absorption wavelength of 188bet asian silver NPs colloid went up to 450nm under UV-VIS spectrometer and 188bet asian efficiency of reaction reduced significantly comparing to NaBH4.

Figure 1. Diagram of silver nanoparticle preparation

Figure 2. UV-VIS spectrophotometric and TEM image of silver NPs with Na3Cit
 

Figure 3. UV-VIS spectrum and TEM image of silver NPs with NaBH4

Figure 4. UV-VIS spectra of silver nanoparticle samples (AgNPs 30, 31, 33)

Figure 5. UV-VIS spectra of silver NPs solution samples with concentration ratios stabilised/AgNO3: 0.05; 0.275 and 0.5

Figure 6. Silver NPs solution in different concentrations

Figure 7. UV-VIS spectra of silver nanoparticles AgNPs 27 at concentrations of 0.1 , 0.3 , 0.5 , 1 and 1.5

Figure 8. TEM of AgNPs 32

It means that 188bet asian reduction capacity of Ag+ ions to Ago by NaBH is higher than Na Cit. As a result, NaBH was comparing to AgNO3. Figure 5 shows 188bet asian UV-VIS spectrographic chromatograms of silver NPs colloids with NaBH4 to AgNO3 ratios being 2:1; 3:1; 4:1 at 0.380; 0.900; 0.683 peak heights respectively. It means that 188bet asian content of reducing agents would considerably affect 188bet asian numbers of silver NPs produced (Figure 4). 188bet asian results showed that 188bet asian highest concentration of silver NPs colloid was chosen for 188bet asian synthesis of silver NPs.

    Silver nanoparticle preparation conditions

-    Effect of NaBH4/AgNO3 concentration ratio
In order to achieve 188bet asian highest reaction efficiency, 188bet asian NaBH4 reductant must be used in excess amount obtained at NaBH4 to AgNO3 ratio of 3:1.

-    Effect of 188bet asian stabilising agent/AgNO3 ratio

188bet asian results in Figure 5 indicated that 188bet asian UV-VIS absorption peak height of products obtained from a 0.05 ratio of stabilising agent to AgNO3 is much lower than that from 0.275 and 0.5 ratios. It can be explained that too low stabilising agent concentration  will  not create mixed molecular surrounding silver NPs, and this is 188bet asian reason why 188bet asian nanoparticle size increases and 188bet asian number of silver NPs decreases. 188bet asian highest UV-VIS absorption peak was obtained from 188bet asian 0.5 ratio of stabilising agent to AgNO3.

    Evaluation of silver NPs colloid characteristics

Silver NPs colloids were prepared at  different concentrations from 0.2 to 2,000ppm (Figure 6). UV-VIS absorption spectrometer of range of silver NPs concentrations was shown in Figure 7. 188bet asian diluted samples all reached a peak of 400nm which indicated that 188bet asian properties of silver NPs were not affected by 188bet asian dilution in water.

188bet asian TEM image of silver NPs at 2,000ppm (Figure 8) showed that 188bet asian size of silver NPs was at approximately 15 ± 4nm. These nanoparticles meet 188bet asian requirements of size which is effective for biocide (< 100nm).

Figure 9 showedtheresultsofsizedistribution of silver NPs at concentration, 2000ppm, and 188bet asian mean size is around 19nm.

    Isolation and identification of bacteria

188bet asian EPC-KK2 sulfate reducing strain was isolated from oil sample MSP10-1014. On 188bet asian microscope, these cells are short rod shaped, Gram-negative bacteria, planktonic. This type of bacteria is anaerobic, and anaerobic respiration uses sulfate as 188bet asian final electron acceptor, producing H2S. They grow on lactate substrates. Optimum conditions of growth include: appropriate temperature is 30 - 32oC; pH is 7.5; and salt concentration of NaCl is 2.5g/l.

188bet asian EPC-KK2 strain was classified and identified by analysing and sequencing 16S rRNA genes.

188bet asian    results    of    gene    analysis    (Figure 10) of EPC-KK2 showed that 188bet asian amplified genomes were similar to Desulfomicrobium baculatum KF536747.1 (Figure 11). From 188bet asian results of sequencing 16S rRNA genes and  188bet asian cell   morphology   (Figure   12),   EPC-KK2   can be identified as Bacteria; Proteobacteria; Deltaproteobacteria; Desulfovibrionales; Desulfomicrobiaceae; Desulfomicrobium baculatum.

Figure 9. Size distribution of AgNPs 27 in solution

Figure 10. Sequence genes of strain EPC-KK2

Figure 11. Phylogenetic position of EPC-KK2 species with relatives based on16S rRNA sequences General characteristics of 188bet asian bacterium Desulfomicrobium baculatum:

Cells: rod or  ellipsoidal-shaped  cells, 0.5 - 0.9 x 1.3 - 2.9μm, with round ends, either singly or in pairs [10]. Gram- negative stain reaction and having cell- wall structure. Cells are motile, usually by a single polar flagellum. Endospores are not formed.

Growth condition: anaerobic, pre- reduced medium or reducing agent required in medium for growth. Growth can occur by anaerobic respiration with sulfate or sulfoxy-anions as terminal electron acceptor, producing H2S. 
Optimal temperature from 25 - 30oC.

Substrates used: simple organic compounds that serve as electron donors during sulfate respiration include lactate, pyruvate,  ethanol,  formate  and hydrogen. Sulfate respiration with lactate as electron donors is incomplete, with 188bet asian formation of acetate and CO2. Hydrogenase is present. Cells  contain  b- and c-type cytochromes. Metabolism can also be fermentative on simple organic substrates, including pyruvate, malate or fumarate; carbohydrates are not fermented. No specific vitamins are required [10].

    Antibacterial effect of silver NPs

There are a number of theories on 188bet asian antibacterial ability of silver NPs colloid, in which 188bet asian absorbed theory is most convincing. 188bet asian main idea of 188bet asian theory is that 188bet asian bacterial cell is inactive due to 188bet asian electron storage between 188bet asian negative charged surface of 188bet asian cell and 188bet asian Ag+ ions which have been absorbed to it. These ions would penetrate into 188bet asian inside of 188bet asian bacterial cell and inactivate them. 

Table 1. 188bet asian effect of NPs size and concentration of AgNPs colloids on EPC-KK2

Table 2. Test on antibacterial concentrations of AgNPs 25 with EPC-KK2

Table 3. Test on antibacterial concentrations of AgNPs 27 with EPC-KK2

188bet asian silver NPs samples  collected  are  tested  with  188bet asian antibacterial effect based on their particle sizes. According to announced  studies,  188bet asian  size  of  silver  NPs is an important factor that affects 188bet asian antibacterial property. For different size of silver NPs, 188bet asian activities of 188bet asian solution on 188bet asian bacteria are very different [8]. Overall, 188bet asian size of 188bet asian silver NPs that is effective for bacteria is within 188bet asian range of 1 to 50nm. For 188bet asian isolated strain,    188bet asian size of silver NPs is within 1 - 50nm. 188bet asian effective concentration of silver NPs solution on different bacteria varies. Normally, 188bet asian  minimum  inhibited  concentration of silver NPs solution is about 10 - 75ppm. 188bet asian silver NPs solutions which are prepared and tested in 188bet asian study include: AgNPs 25, AgNPs 27, AgNPs 31, and AgNPs 34. These solutions did not show precipitation and were tested with 188bet asian EPC-KK2 type. 188bet asian results are shown in Table 1.

Figure 12. Sulfate reducing bacteria EPC-KK2 under Leica DM750 microscope

Table 1 shows 188bet asian ability of silver NPs colloid AgNPs 25 and AgNPs 27 to kill EPC-KK2 strain with 188bet asian mean particle size < 25nm. AgNPs 31 and AgNPs 34 with 188bet asian mean particle size > 25nm did not kill EPC-KK2 strain, effectively.

With 188bet asian silver NPs size < 25nm, 188bet asian antibacterial effect of AgNPs 25 and AgNPs 27 were observed at different concentration between 0 - 30ppm. 188bet asian results in Tables 2 and 3 show that EPC-KK2 was not observed after 188bet asian experiment with AgNPs 25 and AgNPs 27 samples at 188bet asian concentration of 20ppm.

Figure 13. EPC-KK2 strain grown again in test of silver NPs

Figure 14. EPC-KK2 strain killed in test of silver NPs

Pictures of 188bet asian cultivated EPC-KK2 strain in 188bet asian experiment to assess 188bet asian antibacterial ability of silver NPs colloid are shown in Figures 13 and 14.
188bet asian results of 188bet asian  tests  show  that  188bet asian antibacterial ability of AgNPs 27 and AgNPs 25 samples are highly effective at 188bet asian concentration of 20ppm for 188bet asian EPC- KK2 strain.
4.    Conclusion
188bet asian EPC-KK2 strain, which was isolated from 188bet asian Bach Ho crude oil samples and classified as Desulfomicrobium baculatum, was used to evaluate 188bet asian antibacterial effectiveness of silver NPs colloid. Silver NPs (mean silver NPs size 18 -  21nm)  were effective to reduce 188bet asian antibacterial activity from 106tb/ml to < 10tb/ml (99%) at 20ppm. 188bet asian results showed that silver NPs are highly effective in killing SRB. This would create a new approach for applying silver NPs as biocide in Vietnam's oil and gas industry. References
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