A method for simultaneously removing antibiotic-resistant bacteria and drug-resistant genes by combining interface heat-fenton oxidation driven by sunlight

Abstract

This invention relates to a method for the simultaneous removal of antibiotic-resistant bacteria and resistance genes using a solar-driven interfacial heat-Fenton oxidation-like process. The method involves placing a photothermal film loaded with carbon black composite photothermal material in a photothermal sterilization reactor, introducing antibiotic-resistant wastewater, adding H2O2, turning on a xenon lamp to preheat the photothermal film, sealing the reactor, and allowing the wastewater to pass through the photothermal film under gravity. This invention utilizes a photothermal conversion material to absorb light radiation and convert it into thermal radiation, treating antibiotic-resistant bacteria through the high-temperature interfacial heat effect generated on the photothermal film. High-temperature interfacial heat causes localized heating of the bacterial cell membrane structure, leading to a certain degree of breakage of fatty acid chains in the bacterial membrane lipid bilayer. This increases cell membrane permeability, and the resulting physical damage damages the bacterial outer membrane structure, further increasing membrane permeability and thus disrupting membrane integrity.

Description

Technical Field

[0001] This invention relates to a method for the simultaneous removal of antibiotic-resistant bacteria and resistance genes using a solar-driven interfacial thermal-Fenton-like oxidation process, belonging to the fields of chemistry and environmental technology for antibiotic wastewater treatment. Background Technology

[0002] With the widespread use of antibiotics in medicine, animal husbandry, and aquaculture, antibiotic residues and the resulting antibiotic-resistant bacteria (ARBs) and antibiotic resistance genes (ARGs) pose a global threat to the ecological environment. Antibiotic wastewater mainly originates from pharmaceutical factories, hospitals, and domestic sewage. If this wastewater is discharged into the environment without effective treatment, it will not only pollute water bodies but also lead to the spread of antibiotic-resistant bacteria and the horizontal transfer of antibiotic resistance genes, thereby exacerbating antibiotic resistance and the emergence of "superbugs." Currently, traditional technologies for treating antibiotic wastewater mainly include physical, chemical, and biological methods. Physical methods can effectively remove a certain amount of antibiotics from water through physical processes such as adsorption and membrane separation, but their effectiveness in removing antibiotic-resistant bacteria and antibiotic resistance genes is very limited. While physical methods can remove some resistant bacteria, they are ineffective in removing resistance genes from biofilms or inside cells. While chemical methods such as advanced oxidation processes (AOPs) and ozonation can disrupt the molecular structure of antibiotics and oxidants are highly efficient at removing extracellular resistance genes from bacteria, they still struggle to effectively contact internal resistance genes. Therefore, treating drug-resistant bacteria and resistance genes requires high-dose chemical reagents to achieve effective inactivation and degradation. Biological methods, such as activated sludge processes and biofilters, utilize microbial physiological processes to degrade antibiotics with stable efficiency, but they suffer from long treatment cycles, complex preparation, and the potential for introducing new resistance patterns. Therefore, developing a highly efficient method for removing antibiotic-resistant bacteria and resistance genes from antibiotic wastewater is crucial for addressing the current problems in antibiotic wastewater treatment.

[0003] Photothermal sterilization technology utilizes the photothermal effect to kill microorganisms. By exposing the target object to a light source, the material's photothermal conversion properties transform light energy into heat energy, achieving the effect of killing microorganisms. It features rapid elimination of target microorganisms, no secondary pollution, and low cost, but its destructive effect on drug-resistant genes is limited. Fenton advanced oxidation technology is a commonly used process in wastewater treatment plants. Based on the highly oxidizing free radicals in the Fenton reaction, it can non-selectively attack and mineralize most organic pollutants, achieving highly efficient removal in a short time. Studies have also shown that it has a good degradation and destruction effect on gene molecules. However, its effect on antibiotic-resistant bacteria is not ideal because the biofilm structure of antibiotic-resistant bacteria can resist external attacks. Low doses of Fenton reagent have limited destructive effect on the membrane structure, and a certain reaction time may be required to inactivate drug-resistant bacteria. Antibiotic resistance genes within bacteria can be transmitted between different bacteria through horizontal gene transfer, as genetic information. Even if resistant bacteria are inactivated, low-dose Fenton reagent is not effective enough in reducing resistance genes, and they may still remain in the environment to continue spreading resistance. However, blindly using high-dose Fenton reagent will increase the cost of the process and generate more iron sludge.

[0004] In summary, given the shortcomings of existing photothermal sterilization technology and Fenton treatment process, there is an urgent need to develop new technologies to overcome these deficiencies, thereby improving the treatment efficiency of ARBs and ARGs and saving treatment costs. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for the simultaneous removal of antibiotic-resistant bacteria and resistance genes using a combination of sunlight-driven interfacial heat and Fenton-like oxidation.

[0006] This invention is achieved through the following technical solution.

[0007] A method for simultaneously removing antibiotic-resistant bacteria and resistance genes using a solar-driven interfacial heat-Fenton-like oxidation process includes the following steps:

[0008] (1) Place the photothermal film loaded with carbon black composite photothermal material in a photothermal sterilization reactor, introduce antibiotic-resistant bacteria wastewater into the photothermal sterilization reactor through the inlet, add H2O2 solution to control the concentration of the reaction system at 10-100 mmol / L, and stir thoroughly.

[0009] (2) A xenon lamp light source is arranged above the photothermal film loaded with carbon black composite photothermal material. The xenon lamp light source is turned on to preheat the photothermal film. The photothermal disinfection reactor is sealed. Under the action of gravity, the wastewater passes through the photothermal film. During the process of passing through the film, photothermal-Fenton-like oxidation disinfection treatment is continuously carried out to remove antibiotic-resistant bacteria and drug-resistant genes. The treated wastewater is collected into the liquid collection column and discharged through the outlet.

[0010] According to a preferred embodiment of the present invention, in step (1), the photothermal film loaded with carbon black composite photothermal material is prepared by the following method:

[0011] Carbon black composite photothermal material powder and sodium dodecylbenzenesulfonate were dissolved in ultrapure water at a mass ratio of 1:10 and sonicated for 2 hours to obtain mixed solution a. Polyvinyl alcohol solution was added to the mixed solution to make the mass ratio of carbon black composite photothermal material powder to PVA 2:1. The mixture was then sonicated for 1 hour to mix evenly. The sonicated mixed solution b was deposited onto a 0.45 μm PTFE microfiltration membrane using an ultrafiltration device. The membrane was dried in a vacuum drying oven at 60℃ for 40 min and then naturally dried for 12 h. The membrane was rinsed with ultrapure water to remove chemical residues, resulting in a photothermal film loaded with carbon black composite photothermal material.

[0012] According to a preferred embodiment of the present invention, the volume concentration of the polyvinyl alcohol solution is 2%.

[0013] According to a preferred embodiment of the present invention, the carbon black composite photothermal material is an Fe / Mn / carbon black composite photothermal material.

[0014] According to a preferred embodiment of the present invention, the Fe / Mn / carbon black composite photothermal material is prepared by the following method:

[0015] Add 1-5g of FeCl3·6H2O, 1-5g of MnCl2·4H2O, and 3-6g of carbon black powder to 50mL of ethylene glycol solution and stir for 60min. Transfer the resulting mixture to a stainless steel autoclave lined with polytetrafluoroethylene and heat it in an oven at 200℃ for 15h. After heating, centrifuge to collect the precipitate and wash it twice each with ethanol and deionized water. Then, vacuum dry it at 70℃ for 20h and finally calcine it at 500℃ for 2h in a N2 atmosphere to obtain Fe / Mn / carbon black composite photothermal material powder.

[0016] According to a preferred embodiment of the present invention, the photothermal sterilization reactor includes a reactor body with an open top, a support frame at the bottom of the reactor body, a photothermal film loaded with carbon black composite photothermal material on the support frame, a xenon lamp light source at the top of the photothermal film, a liquid collection column at the bottom of the support frame, the liquid collection column being connected to a liquid outlet on the side wall of the reactor body through a channel, a liquid inlet on the side wall of the reactor body above the photothermal film, and a quartz glass cover at the opening of the reactor body.

[0017] According to a preferred embodiment of the present invention, in step (2), the power of the xenon lamp is 250-350W, the wavelength is 200-2000nm, and an AM 1.5G filter is used to correct the solar spectrum.

[0018] According to a preferred embodiment of the present invention, in step (2), the preheating time is 10-30 min.

[0019] According to a preferred embodiment of the present invention, in step (2), the xenon lamp light source is 15-30 cm away from the photothermal film.

[0020] Compared with existing technologies, the technical features and advantages of this invention are as follows:

[0021] 1. This invention employs a solar-driven photothermal-Fenton-like oxidation combined process to remove antibiotic-resistant bacteria and antibiotic resistance genes from wastewater. The photothermal conversion material absorbs light radiation and converts it into heat radiation, which is then used to treat antibiotic-resistant bacteria through a high-temperature interfacial heat effect generated on the photothermal film. This high-temperature interfacial heat causes localized heating of the bacterial cell membrane structure, leading to a certain degree of breakage of fatty acid chains in the bacterial membrane lipid bilayer. This increases cell membrane permeability, and the resulting physical damage damages the bacterial outer membrane structure, further increasing membrane permeability and thus disrupting membrane integrity. The high temperature also causes protein denaturation on the membrane, causing bacteria to lose their normal physiological and biochemical functions, resulting in bacterial inactivation. Compared to simply increasing the dosage of chemical reagents to intensify membrane structure destruction, photothermal sterilization does not cause secondary pollution and can effectively inactivate bacteria while reducing environmental impact.

[0022] 2. The method of this invention utilizes the characteristic that bacteria are induced to produce reactive oxygen species (ROS) when exposed to heat. It works in conjunction with the external heat effect and Fenton-like oxidation to further attack and destroy the bacterial membrane, accelerate the release of drug-resistant genes that are difficult to remove from the inside into the external environment, and more quickly come into contact with the strong oxidizing free radicals generated by the external Fenton-like reaction. This makes the drug-resistant gene fragments destroyed and degraded more quickly, improves the treatment efficiency of Fenton-like oxidation, and thus reduces the amount of Fenton reagent used and controls the treatment cost.

[0023] 3. The method of the present invention utilizes gravity to allow wastewater to pass through the photothermal membrane, reducing the cost of external force required for traditional membrane processing. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the photothermal sterilization reactor used in this invention;

[0025] In the diagram, 1 is the xenon lamp light source; 2 is the quartz glass cover; 3 is the liquid inlet; 4 is the wastewater; 5 is the photothermal film; 6 is the liquid collection column; and 7 is the liquid outlet.

[0026] Figure 2 This is a comparison chart of the inactivation effects of amoxicillin-resistant bacteria under different experimental conditions described in Examples 1, 2, 3, and 4 of the present invention. The horizontal axis AE represents Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, and Example 1, respectively.

[0027] Figure 3 This is a comparison chart of the effects of amoxicillin antibiotic resistance gene reduction under different experimental conditions described in Examples 1, 2, 3, and 4 of the present invention. The horizontal axis AE represents Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, and Example 1, respectively.

[0028] Figure 4 This is a comparison chart of the changes in malondialdehyde (MDA) content in amoxicillin-resistant bacteria under different experimental conditions described in Examples 1, 2, 3, and 4 of the present invention. The horizontal axis AE represents Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, and Example 1, respectively.

[0029] Figure 5 This is a comparison chart of the changes in ATP content of amoxicillin-resistant bacteria under different experimental conditions described in Examples 1, 2, 3, and 4 of the present invention. The horizontal axis AE represents Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, and Example 1, respectively. Detailed Implementation

[0030] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0031] Example 1

[0032] Preparation of Fe / Mn / carbon black composite photothermal material:

[0033] Add 2g of FeCl3·6H2O, 3g of MnCl2·4H2O, and 5g of carbon black powder to 50mL of ethylene glycol solution and stir for 60min. Transfer the resulting mixture to a stainless steel autoclave lined with polytetrafluoroethylene and heat it in an oven at 200℃ for 15h. After heating, centrifuge to collect the precipitate and wash it twice each with ethanol and deionized water. Then, vacuum dry it at 70℃ for 20h and finally calcine it at 500℃ for 2h in a N2 atmosphere to obtain Fe / Mn / carbon black composite photothermal material powder.

[0034] Preparation of photothermal thin films:

[0035] Fe / Mn / carbon black composite photothermal material powder and sodium dodecylbenzenesulfonate were dissolved in ultrapure water at a mass ratio of 1:10 and ultrasonicated for 2 hours to obtain mixed solution a. Polyvinyl alcohol solution was added to the mixed solution to make the mass ratio of carbon black composite photothermal material powder to PVA 2:1. The mixture was then ultrasonicated for 1 hour to mix evenly. The ultrasonicated mixed solution b was deposited onto a 0.45 μm PTFE microfiltration membrane using an ultrafiltration device. The membrane was dried in a vacuum drying oven at 60℃ for 40 min and then naturally dried for 12 h. The membrane was rinsed three times with ultrapure water to remove chemical residues, resulting in a photothermal film loaded with carbon black composite photothermal material.

[0036] A method for simultaneously removing antibiotic-resistant bacteria and resistance genes using a solar-driven interfacial heat-Fenton-like oxidation process includes the following steps:

[0037] (1) The prepared photothermal film loaded with carbon black composite photothermal material is placed in a photothermal sterilization reactor, and 20 ml of ~10 ml of water is introduced into the photothermal sterilization reactor through the inlet. 7 For antibiotic-resistant bacteria wastewater with a concentration on the order of CFU / mL, add H2O2 solution to control the concentration of the reaction system at 30 mmol / L, and stir thoroughly.

[0038] (2) Place a xenon lamp light source above the photothermal film loaded with carbon black composite photothermal material, adjust the light source to be 20 cm above the photothermal film placed in the reactor in step (1), turn on the xenon lamp light source, preheat the photothermal film for 20 min, cover it with a quartz glass cover to maintain a relatively sealed system, and the wastewater passes through the photothermal film under the action of gravity. During the process of passing through the film, photothermal-Fenton oxidation disinfection treatment is continuously carried out to remove antibiotic resistant bacteria and drug resistance genes at the same time. The treated wastewater is collected into the liquid collection column and discharged through the outlet.

[0039] The photothermal sterilization reactor includes a reactor body with an open top, a support frame at the bottom of the reactor body, a photothermal film 5 loaded with carbon black composite photothermal material on the support frame, a xenon lamp light source 1 on the upper part of the photothermal film 5, a liquid collection column 6 below the support frame, the liquid collection column 6 being connected to the liquid outlet 7 on the side wall of the reactor body through a channel, a liquid inlet 3 on the side wall of the reactor body above the photothermal film, and a quartz glass cover 2 at the opening of the reactor body.

[0040] Comparative Example 1

[0041] The method for simultaneously removing antibiotic-resistant bacteria and resistance genes using a combination of sunlight-driven interfacial heat and Fenton-like oxidation, as described in Example 1, differs in that:

[0042] No photothermal material is loaded on the photothermal film; a light source is used; otherwise, it is exactly the same as in Example 1.

[0043] Comparative Example 2

[0044] The method for simultaneously removing antibiotic-resistant bacteria and resistance genes using a combination of sunlight-driven interfacial heat and Fenton-like oxidation, as described in Example 1, differs in that:

[0045] The photothermal film does not have any photothermal material loaded on it, and no light source is used. It only provides a thermal environment with the same system temperature. Everything else is exactly the same as in Example 1.

[0046] Comparative Example 3

[0047] The method for simultaneously removing antibiotic-resistant bacteria and resistance genes by combining sunlight-driven interfacial heat and Fenton-like oxidation as described in Example 1 differs from that in Example 1 in that no photothermal material is loaded on the photothermal film, a thermal environment with the same system temperature is provided, and a light source is used. Otherwise, it is exactly the same as Example 1.

[0048] Comparative Example 4

[0049] The method for simultaneously removing antibiotic-resistant bacteria and resistance genes by combining sunlight-driven interfacial heat and Fenton-like oxidation as described in Example 1 is identical to that in Example 1 except that no light source is used.

[0050] Experimental Example

[0051] The photothermal-Fenton-like oxidation disinfection experiment was conducted on wastewater containing amoxicillin-resistant bacteria using the protocol of Example 1. The initial solution volume was 20 mL, and the H2O2 concentration was 30 mmol / L. The same parameters were applied to Comparative Examples 1-4 for disinfection of amoxicillin-resistant bacterial wastewater. During the photothermal-Fenton-like oxidation disinfection process, 1 mL samples were taken at fixed times (0 min, 5 min, 15 min, 30 min, and 40 min) to confirm the inactivation of resistant bacteria using the plate coating method. A 1 mL sample was taken at 2 h to detect the reduction of resistance genes. After the experiment, the inactivation of antibiotic-resistant bacteria, the reduction of resistance genes, and membrane damage were tested. Figures 2-5 .

[0052] 1. Through Figure 2It can be seen that Example 1 has a more significant inactivation effect on drug-resistant bacteria compared to Comparative Examples 1-4. During the disinfection process within 40 minutes, the inactivation effect of drug-resistant bacteria in Comparative Examples 1-4 remained at 3-4 orders of magnitude, while Example 1 achieved an inactivation effect of 7 orders of magnitude. The photothermal-Fenton-like oxidation combined process in the reaction system of Example 1 is complete, while mechanisms involving only light action (Comparative Example 1), only heat action (Comparative Example 2), only photothermal action (Comparative Example 3), and only Fenton-like oxidation (Comparative Example 4) have limited effectiveness in inactivating drug-resistant bacteria. In Example 1, on the one hand, the photothermal material efficiently and rapidly absorbs light energy and converts it into heat energy, generating a high-temperature thermal effect on the surface of the photothermal film. This heat effect comes into contact with drug-resistant bacteria, causing a certain degree of damage to the lipids and proteins in the bacterial membrane structure, altering the cell membrane permeability and leading to membrane structure destruction, thus preventing the normal functioning of bacteria. On the other hand, the Fe / Mn metal components in the photothermal material react with H2O2 to form a Fenton-like system, promoting the generation of highly oxidizing free radicals. This also oxidizes and attacks cell membrane structural components, accelerating membrane structure destruction in conjunction with the high-temperature photothermal effect. Therefore, the photothermal-Fenton-like oxidation combined process employed in this invention achieves a more significant effect in inactivating drug-resistant bacteria.

[0053] 2. Through Figure 3 It can be seen that Example 1 has a higher degree of resistance gene reduction compared to Comparative Examples 1-4. This is because under the combined photothermal-Fenton-like oxidation process, the bacterial membrane structure is more severely damaged. The resistance gene genetic material that is difficult to remove from the cell is released into the extracellular space at a faster rate and at a higher content within the same time (2h) compared to Comparative Examples 1-4, which is conducive to Fenton-like oxidation destroying the resistance gene fragment.

[0054] 3. To verify that Example 1 showed a more significant degree of bacterial membrane structure damage compared to Comparative Examples 1-4, the changes in malondialdehyde (MDA) content and ATP content were tested. Studies have shown that MDA is a product of lipid structure oxidation and decomposition. Impaired membrane permeability and loss of inorganic phosphates through the membrane during intracellular nucleotide efflux lead to ATP hydrolysis and a decrease in its content. Specifically, in Example 1, compared to Comparative Examples 1-4, the MDA content significantly increased at each time point, while the ATP content decreased to some extent. This indicates that the photothermal-Fenton-like oxidation combined process in this invention has a significant destructive effect on the bacterial membrane structure.

[0055] Therefore, this invention utilizes the high-temperature interfacial thermal effect of a photothermal thin film in a 30 mmol / L H2O2 system to synergistically treat antibiotic-resistant bacterial wastewater using Fenton-like oxidation, achieving excellent bacterial inactivation and resistance gene reduction during disinfection. Compared to simply increasing the concentration of chemical oxidants to enhance bactericidal activity and reduce resistance genes, this method reduces the consumption and control costs of Fenton reagent.

[0056] Example 2

[0057] The method for simultaneously removing antibiotic-resistant bacteria and resistance genes using a combination of sunlight-driven interfacial heat and Fenton-like oxidation, as described in Example 1, differs in that:

[0058] The concentration of H2O2 in the reaction system was 10 mmol / L, and the order of magnitude of antibiotic-resistant bacteria was ~10. 6 CFU / mL.

[0059] Example 3

[0060] The method for simultaneously removing antibiotic-resistant bacteria and resistance genes using a combination of sunlight-driven interfacial heat and Fenton-like oxidation, as described in Example 1, differs in that:

[0061] The concentration of H2O2 in the reaction system was 50 mmol / L, and the order of magnitude of antibiotic-resistant bacteria was ~10. 8 CFU / mL.

[0062] Example 4

[0063] The method for simultaneously removing antibiotic-resistant bacteria and resistance genes using a combination of sunlight-driven interfacial heat and Fenton-like oxidation, as described in Example 1, differs in that:

[0064] The concentration of H2O2 in the reaction system was 70 mmol / L, and the order of magnitude of antibiotic-resistant bacteria was ~10. 9 CFU / mL.

Claims

1. A method for simultaneously removing antibiotic-resistant bacteria and resistance genes using a solar-driven interfacial thermal-Fenton-like oxidation process, comprising the following steps: (1) Place the photothermal film loaded with carbon black composite photothermal material in the photothermal sterilization reactor, introduce antibiotic resistant bacteria wastewater into the photothermal sterilization reactor through the water inlet, add H2O2 solution to control the concentration of the reaction system at 10~100mmol / L, and stir thoroughly. The photothermal thin film loaded with carbon black composite photothermal material is prepared by the following method: Carbon black composite photothermal material powder and sodium dodecylbenzenesulfonate were dissolved in ultrapure water at a mass ratio of 1:10 and ultrasonicated for 2 hours to obtain mixed solution a. Polyvinyl alcohol solution was added to the mixed solution to make the mass ratio of carbon black composite photothermal material powder to PVA 2:

1. Then, the mixture was ultrasonicated for 1 hour to mix evenly. The ultrasonicated mixed solution b was deposited onto a 0.45 μm PTFE microfiltration membrane using an ultrafiltration device. The membrane was dried in a vacuum drying oven at 60 ℃ for 40 min and then naturally dried for 12 h. The membrane was rinsed with ultrapure water to remove chemical residues and obtain a photothermal film loaded with carbon black composite photothermal material. The volume concentration of the polyvinyl alcohol solution is 2%, and the carbon black composite photothermal material is an Fe / Mn / carbon black composite photothermal material, which is prepared by the following method: Add 1-5g of FeCl3·6H2O, 1-5g of MnCl2·4H2O, and 3-6g of carbon black powder to 50 mL of ethylene glycol solution and stir for 60 min. Transfer the resulting mixture to a stainless steel autoclave lined with polytetrafluoroethylene and heat it in an oven at 200℃ for 15 h. After heating, centrifuge to collect the precipitate and wash it twice each with ethanol and deionized water. Then, vacuum dry it at 70℃ for 20 h and finally calcine it at 500℃ for 2 h in a N2 atmosphere to obtain Fe / Mn / carbon black composite photothermal material powder. (2) A xenon lamp light source is arranged above the photothermal film loaded with carbon black composite photothermal material. The power of the xenon lamp is 250-350W and the wavelength is 200~2000 nm. An AM 1.5G filter is used to correct the solar spectrum. The xenon lamp light source is turned on to preheat the photothermal film. The photothermal disinfection reactor is sealed. Under the action of gravity, the wastewater passes through the photothermal film. During the process of passing through the film, photothermal-Fenton-like oxidation disinfection treatment is continuously carried out to remove antibiotic resistant bacteria and drug resistance genes. The treated wastewater is collected in the liquid collection column and discharged through the outlet.

2. The method according to claim 1, characterized in that, The photothermal sterilization reactor includes a reactor body with an open top, a support frame at the bottom of the reactor body, a photothermal film loaded with carbon black composite photothermal material on the support frame, a xenon lamp light source above the photothermal film, a liquid collection column below the support frame, the liquid collection column being connected to a liquid outlet on the side wall of the reactor body through a channel, a liquid inlet on the side wall of the reactor body above the photothermal film, and a quartz glass cover at the opening of the reactor body.

3. The method according to claim 1, characterized in that, In step (2), the preheating time is 10-30 minutes.

4. The method according to claim 1, characterized in that, In step (2), the xenon lamp light source is 15-30cm away from the photothermal film.

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