Chapter 6. Inactivation of the E. coli and the B. subtilis Using Air-Based
6.4 Summary of Chapter 6
Major findings of the study of inactivation of E. coli and B. subtilis using the developed planar air-based DBD APPJ system are summarized as follows:
(1) The APPJ was used to inactivate E. coli (Gram negative) and B. subtilis (Gram positive) up to 107 CFU/mL using the post-discharge region. Results show that the post-discharge jet region is very efficient in inactivating these two bacteria as previous studies using the discharge region, should the working gas contains appreciable oxygen addition, which in turn generates abundant ozone.
(2) In addition, the inactivation is more effective by compressed-air APPJ compared to that by oxygen APPJ, possibly through the assistance of nitrous oxide existing in the former. Major advantage by using post-discharge jet region is its flexibility in practical applications, in which the DBD becomes a stand-alone module that can be used to treat essentially any sample as compared to the conventional applications by using the discharge region.
Chapter 7
Sterilization of the B. subtilis Spore Using Air/Carbon Fluorine DBD APPJ
In this chapter, sterilization of the B. subtilis spore using the developed planar air-carbon fluorine DBD APPJ system has been described in detail. Details of the experimental setup and characteristics of the discharge have been shown in Chapter 2 and Chapter 3, respectively, and are skipped here for brevity. XPS analysis of PP film treated by the air-carbon fluorine DBD APPJ, direct visualization of the Petri dish with bacteria inside and SEM images before and after plasma jet treatment are described in the following in turn. Some important findings have been summarized at the end of this Chapter.
7.1 XPS Analysis of PP Film
Since it is difficult to directly probe the existence of the F related atoms or compounds in the discharge and post-discharge jet regions using OES data, we have instead verified the existence of chemically active F radicals indirectly. We have applied the air-carbon fluorine DBD APPJ to treat a PP film and examined the surface composition using XPS analysis. Table 7-1 summarizes the measured chemical composition of the PP film surface using XPS analysis and the measured CAs after plasma jet treatment. Clearly, the O/C ratio increased dramatically from 0.30 (untreated) to 0.40 (z=14 mm for 2% CF4/air oxygen cases), which was similar to the result in [Kim et al., 2006]. We have to admit that the measured trace amount F atoms in the PP film surface is obviously smaller than the experimental uncertainties of measurement..
However, the results clearly showed that some carbon atoms were effectively removed by the plasma jet, which could be only caused by the F related species since the O/C ratio obtained by the use of pure air discharge is smaller (0.35) than 0.40. Of course, further study is required to unveil the role of F related radicals and compounds in the sterilization of bacteria.
7.2 Appearance of Petri Dish for B. subtilis Spore
In the current study, the number of B. subtilis spores on each petri dish was controlled at ~105, 106 and 107 spore/mL before plasma jet treatment. Figures 7-1 and 7-2 show the appearance of the B. subtilis spore after incubation with different designated bacterial numbers (i.e., spore/mL) and times (i.e., numbers of passes) by the air plasma and CF4/air (2%) plasma jets, respectively. It was found that the CF4/air (2%) APPJ treatment resulted in the efficient inactivation of the B. subtilis spores after 10 passes (residence time: 1.0 s) exposures for treatment distances (14 mm).
Tables 7-2 and 7-3 summarize the survival rates (%) of the B. subtilis spores under various test conditions (number of bacteria and number of passes) air and CF4/air (2%), respectively. The results clearly showed that the discharges containing Carbon Fluorine (CF4) performed excellently in activating the B. subtilis spores because of the existence of F atoms that are very chemically active in etching with the spores [Lerouge et al., 1999].
7.3 SEM Images of Untreated and Treated B. subtilis Spore
The SEM images of untreated and treated B. subtilis spore using a CF4/air (2%) plasma jet are shown in Figure 7-3. The B. subtilis spore underwent some
morphological erosion as compared to the control dish. The bacteria were actually sterilized based on our experimental observation (see Tables 7-2 and 7-3). This was similar to polymer material etching studies which showed clear ruptured bacterium surfaces after plasma treatment. This definitely deserves future investigation (e.g., using bio-TEM) to clarify the underlying cause for the efficient sterilization as found in the current study.
7.4 Summary of Chapter 7
Major findings of the study of sterilization of B. subtilis spore using the developed planar air-carbon fluorine DBD APPJ system are summarized as follows:
(1) In the PP film treated by CF4/air (2%) DBD plasma, the O/C ratio of the XPS data showed an increase as compared with treated by air DBD. This showed that chemically active F related radicals existed in the discharge.
(2) The sterilization of the B. subtilis spore treated by CF4/air (2%) DBD plasma was very inefficient as compared with that treated by air DBD plasma.
Indeed, the role of CF4 mixed in the air DBD discharge for sterilization of the B.
subtilis spore requires further investigation.
Chapter 8
Conclusion and Recommendations for Future Study
In this chapter, major findings of the current thesis are summarized in the following in turn as: 1) Characterization of the planar DBD APPJ; 2) Hydrophilic modification of the PP film; 3) Surface cleaning of the ITO glass; 4) Inactivation of E. coli and B.
subtilis and 5) Sterilization of B. subtilis spore. In addition, recommendations for future study are also outlined at the end of this chapter.
8.1 Summaries of the Thesis
8.1.1 Characterization of the Planar DBD APPJ
Characterizations of the proposed planar DBD APPJ system are summarized as follows:
(1) Direct visualization and OES spectra aided to our understanding the changes in plume color.
(2) OES measurements showed that abundant metastble nitrogen was generated in the post-discharge jet region, which was important in later applications.
However, addition of oxygen species reduced the amount of metastable nitrogen tremendously because of reduced plasma density due to electro-negativity of the oxygen.
(3) Gas temperature measurements showed that gas temperatures were low enough in the post-discharge jet region for most of the applications studied in the thesis.
(4) Measurements showed that addition of oxygen enhanced the amount of ozone, which were important in bacteria inactivation.
8.1.2 Hydrophilic Modification of the PP Film
Major findings of the study of hydrophilic modification of the PP film using the developed planar DBD APPJ system are summarized as follows:
(1) The DBD jet was applied to treat the PP film under stationary and non-stationary conditions with various O2/N2 ratios ranging from 0 to 1.6% at different treating distances in the range of 2-20 mm.
(2) Results show that, for stationary PP films, the surface hydrophilic property improves dramatically from 103° (untreated) to a value less than 30° (treated) for contact angles with a wide range of O2/N2 ratios (< 1%) and treating distances (< 10 mm). For the non-stationary PP films, highly hydrophilic surfaces can only be obtained when the PP film is placed near the jet exit (treating distance of 2-4 mm) with an O2/N2 ratio of 0.06-0.2%.
(3) These observations are explained through measured optical emission spectra and ozone concentration data, in which the metastable nitrogen plays a key role in breaking the surface chemical bounds and UV emission (200-300 nm) participates in the process of converting the ozone into oxygen radical. Aging tests show that for stationary PP films the contact angle can still be maintained at ~40° after 24 h, while for the non-stationary test cases it can only be maintained at ~80-90° when the non-stationary speed is near 1 cm/s.
(4) By AFM analysis, we also observed that the less the surface roughness the smaller the contact angle is. Finally, XPS analysis shows that O/C ratio increases dramatically which results from the incorporation of several polar
functional groups containing oxygen into the surface of PP films during plasma treatment. The greater the amount of functional groups C-O and C=O, the better the hydrophilic property of the PP film.
(5) In addition, the number of polar functional groups, such as C=O, having higher binding energy can directly influence the aging time of the hydrophilic property of PP film after the plasma treatment.
8.1.3 Surface Cleaning of the ITO Glass
Major findings of the study of surface cleaning of ITO glass using the developed planar DBD APPJ system are summarized as follows:
(1) Results showed that there existed two distinct regimes having lower CAs in the range of 20-30°. Possible mechanisms of surface cleaning have been presented;
they took into account the spatial distribution of O3, NO-γ UV emission and OES spectra.
(2) For ITO cleaning mechanisms, in the near jet downstream location (z<10 mm), both the metastable N2 [N2(A3
∑
u+)] and ozone photo-induced dissociation played dominant roles in cleaning ITO glass, although their relative importance was unclear and requires further investigation.(3) In the far jet downstream location (z>10mm), when the ratio of O2/N2 was small, only the long-lived metastable N2 [N2(A3
∑
u+)] played a major role in cleaning ITO glass. One final note on using nitrogen as the discharge gas is that nitrogen was easily recycled to a high purity (>99%) using a ceramic membrane [Baker, 2002]8.1.4 Inactivation of E. coli and B. subtilis
Major findings of the study of inactivation of E. coli and B. subtilis using the developed planar air-based DBD APPJ system are summarized as follows:
(1) The APPJ was used to inactivate E. coli (Gram negative) and B. subtilis (Gram positive) up to 107 CFU/mL using the post-discharge region. Results show that the post-discharge jet region is very efficient in inactivating these two bacteria as previous studies using the discharge region, should the working gas contains appreciable oxygen addition, which in turn generates abundant ozone.
(2) In addition, the inactivation is more effective by compressed-air APPJ compared to that by oxygen APPJ, possibly through the assistance of nitrous oxide existing in the former. Major advantage by using post-discharge jet region is its flexibility in practical applications, in which the DBD becomes a stand-alone module that can be used to treat essentially any sample as compared to the conventional applications by using the discharge region.
8.1.5 Sterilization of B. subtilis Spore
Major findings of the study of sterilization of B. subtilis spore using the developed planar air-carbon fluorine DBD APPJ system are summarized as follows:
(1) In the PP film treated by CF4/air (2%) DBD plasma, the O/C ratio of the XPS data showed an increase as compared with treated by air DBD plasma. This showed that chemically active F related radicals existed in the discharge.
(2) The sterilization of the B. subtilis spore treated by CF4/air (2%) DBD plasma was very efficient as compared with that treated by air DBD plasma.
(3) Indeed, the role of CF4 mixed in the air DBD discharge for sterilization of the B.
subtilis spore requires further investigation.
8.2 Recommendations for Future Work
(1) The fluid modeling coupled with a neutral flow solver using detailed plasma chemistry should be conducted to help elucidate the observed physical phenomena, which are otherwise very difficult to understand only based on measurements.
(2) Further tailor of the properties of nitrogen- and air-based discharges using a realistic pulse-based power source with the help of fluid modeling should be conducted to enhance the applicability of the proposed discharges.
(3) For CF4/Air DBD APPJ OES measurement, try to enlarge the relative CFx/F line peak intensity of OES spectrum compare with background (without plasma) CF4/Air. To presume plasma chemistry of the possible existence of CFx/F line peak in discharge region of CF4/Air DBD APPJ.
(4) For CF4/Air DBD APPJ post-discharge region, try to use FTIR measuring reactive F species (as CFx fragment and CF2O) IR spectrum. To presume plasma chemistry of the possible existence of relative F species in post-discharge region of CF4/Air DBD APPJ.
(5) Using ATR-FTIR analysis the bacteria (E. coli and B. subtilis)/B. subtilis spore of surface chemical composition after DBD plasma untreated/treated to explain the cell wall variation of C1s/O1s chemical bonding energy.
(6) For B. subtilis spore sterilization mechanism presuming of CF4/Air DBD APPJ, to study relative references about low pressure CF4/O2 plasma remove photo-resistance mechanism. And try to search the optimum concentration of CF4/Air for B. subtilis spore sterilization.
(7) To use air DBD APPJ treated the similar cell membrane material (liposome bilayers) and compare the SEM of liposome bilayers and E. coli. It can help to
understand plasma chemistry of E. coli outer cell wall.
(8) For PP film treated by nitrogen-based DBD APPJ, the XPS data of chemical composition C and O atom concentration are different with C1s peak curve fitting C-O, C-C, C=O, COO concentration. To discuss with equipment technician about the calculating rules and verify again.
(9) For PP film non-stationary treated by nitrogen-based DBD APPJ case, try to normalize the equation of non-stationary speeds and treatment distances in fixed O2/N2
ratio. It can help to presume PP film contact angle variation in application.
(10) More studies are required to clarify qualitatively and quantitatively what kinds of reactive species for the sterilization of B. subtilis spore using the CF4/air DBD APPJ.
(11) We can possibly apply an enlarged enclosure to cover the post-discharge jet region to form a region of abundant metastable region for some specific applications.
(12) As demonstrated in the present thesis, we have shown that the nitrogen- and air-based DBD APPJ system is very effective with a reduced cost in improving hydrophilic properties and sterilization/inactivation of a polymer surface. It is thus highly promising to apply this APPJ system to improve the cell attachment or bio-compatibility of some bio-materials, for example, PLA ((C6H8O4)n) as the skeleton of artificial vascular graft and PDMS ((C2H6OSi)n) as the microfluidic channels, in a totally dry approach, unlike the conventional chitosan coating ((C6H11NO4)n), which is notoriously time-consuming and very tedious.
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