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Nicotiana tabacum var. Virginia Bio Oil-based Pyrolisis Extraction Have Prominence Antimicrobial Potential Compared to Ethanol Heat Reflux Extraction (EHRE)

Tobacco leaf contains antibacterial secondary metabolite compounds, such as phenol, alkaloids, and essential oils. This study compares the potential antibacterial effects of Indonesian tobacco leaf extracted using the heat reflux method (producing an extract) and pyrolysis method (providing a bio-oil). The tobacco leaf extract was challenged against Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853. The bio-oil from the pyrolysis method showed inhibitory Kirby Bauer zones higher than those of the extract from heat reflux method, with the maximum results in the pyrolysis method indicating zones of 6.35 mm ( S. aureus ), 5.90 mm ( E. faecalis ), 3.97 mm ( E. coli ), and 5.025 mm ( P. aeruginosa ). Further study analyzed the effectiveness of the disc and well diffusion antibacterial test methods for measuring the antibacterial effect of bio-oils against Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853. The bio-oil used in the well diffusion test method showed the most significant antibacterial effectiveness. It showed the biggest inhibition zone, with a maximum of 11.65 mm and 8.90 mm for E. coli and P. aeruginosa . Our results showed Nicotiana tabacum var. Virginia Bio Oil from Ponorogo (Indonesia) is a strong potential antimicrobial, especially using well diffusion test.


Introduction
The risk of nosocomial infection is highest approximately 48 hours after admission to a hospital environment and 30 days after hospital care ends (Suleyman et al. 2018). Such infections can be of exogenous origins, such as from epidemic diseases, person-to-person contact, and contact with devices (Suleyman et al. 2018). Antibiotic resistance caused by inappropriate treatments and therapies can increase the risk of nosocomial infections in patients in hospitals (Gentile et al. 2020). Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, and Pseudomonas aeruginosa are the most common bacteria causing nosocomial infections (Kudo et al. 2014;Iseppi et al. 2020;Jean et al. 2020;Kolar et al. 2020).
The use of antimicrobials from plants for nosocomial infection can reduce the side effects of excessive antibiotics and antibiotic resistance (Cheesman et al. 2017). The Indonesian government's limitation of cigarette production makes it important to look for alternative products, one of which is health products. In addition to social and economic needs, tobacco contains many antibacterial substances and can be used as drugs and antimicrobial health products. One of Indonesia's most common tobacco variants is Nicotiana tabacum var Virginia, which includes 63% of tobacco types in this country (Ahsan 516 Pramono AP et al. et al. 2014). Nicotiana tabacum Var Virginia originated from Virginia (America), brought to Indonesia in 1928, and planted in Bojonegoro for the first time to meet the demand for cigarette tobacco in Indonesia (Pertanian RI 2011). Besides containing high levels of nicotine, pyridine, indole, and d-limonene (Gozan et al. 2014;Andhika Priotomo et al. 2018). Tobacco can be used as an antimicrobial agent because its leaves contain secondary metabolite compounds, including alkaloids, flavonoids, phenols, saponins, and steroids (Sokunvary Oeung et al. 2016;Fauzantoro et al. 2017;Fathi et al. 2018), which can inhibit the growth of bacteria (Pramono et al. 2018).
A common extraction method in extracting antimicrobial compounds of the Nicotiana tabacum is Ethanolic Heat Reflux Extraction (EHRE) methods (Fauzantoro et al. 2017;Pramono et al. 2018;Banožić et al. 2020). Some of the studies used the pyrolysis extraction method to extract the antimicrobial compound of the Nicotiana tabacum (Ario Putra et al. 2019;Fernanda et al. 2021). The pyrolysis method involves heating a substance to a high temperature without the oxygen that can cause the disintegration of chemical compounds, this process results in biooil compounds (Gozan et al. 2014). The advantage of the pyrolisis extraction method is that it can attract active compounds in large amounts, with a high temperature (Andhika Priotomo et al. 2018), but the antimicrobial potential from bio oil product still needs to be evaluated. Here we compared the antimicrobial potential of Nicotiana tabacum L. var. Virginia extracts produced using the heat reflux and pyrolysis methods from Ponorogo, Indonesia. The comparison with another study is also being discussed.

Plant Origin and Sample Collection
Tobacco plants (Nicotiana tabacum L. var. Virginia) as raw material for the extracts were obtained from Ponorogo, East Java Province (Indonesia). Fresh leaves were collected and immediately processed. The tobacco leaves used are tobacco leaves that are approximately three months old and are ready to be harvested. The tobacco plant is a subtropical plant but can grow in low pliers wide climate (29−33°C). Nicotiana tabacum L. var. Virginia is grown in Ponorogo, Indonesia, at the end of the rainy season, and harvested in the dry season, April-Mei in Ponorogo, East Java, Indonesia (Pertanian RI 2011).

Preparation of Leaf Extracts Using the Heat-Reflux Extraction Method (Extract)
Several steps are needed to make an extract using the heat reflux method. First, the tobacco leaves are cleaned with water. Second, the tobacco leaves are dried in an oven at 120°C for 2 hours. After the tobacco leaves are dried, they are ground into a dried leaf powder. Up to 50 g of dried leaf powder is then put into an Erlenmeyer glass and dissolved with 150 ml of ethanol solvent. Then, the solution is heated to a temperature of 80°C. This healing process is a vapour, which is put into a condenser until it forms a concentrated tobacco leaf extract (briefly named extract) (Fauzantoro et al. 2017).

Preparation of Leaf Extracts Using the Pyrolysis Method (Bio-oil)
Another extraction method used is the pyrolysis method. First, the tobacco leaves are cleaned and then dried. After that, the tobacco leaves are chopped into smaller pieces, and 250 g of chopped leaves are converted to a powder form. This is done to improve the efficiency of the pyrolysis process. The pyrolysis reaction is carried out inside a closed pyrolysis reactor so that no external gas can enter the reactor. The tobacco leaves are heated to a temperature of 500°C. The heating process produces gas and char. The gas and char are separated using a cyclone separator. Finally, the gas is cooled until it forms a bio-oil (Gozan et al. 2014).

Bacterial Strains and Growth Conditions
The bacteria used in this study were obtained from the American Type Culture Collection (ATCC). They included the following strains: Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212, Escherichia Coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853. All bacteria were cultured overnight with agitation at 150 rpm, in flasks containing Mueller-Hinton broth (Merck, Darmstadt, Germany), before being used in the experiment.

Antibacterial Activity Assessment
The samples used in this research are divided into several groups: 20%, 40%, 60%, 80%, and 100%. The percentage is obtained by diluting the extract with aquadest or bio-oil with propylene glycol. One hundred percent is the initial concentration obtained from direct extraction results. Then 80%, 60%, 40%, and 20% dilutions are obtained by adding a certain amount. The bio-oils and extracts were then analyzed using Vol. 29 No. 4, July 2022 the Kirby Bauer antibacterial test (Hudzicki 2009;Balouiri et al. 2016). Each experiment used both negative (only with aquadest for EHRE experiment, only with propylene glycol for the pyrolysis extraction experiment) and positive (Ciprofloxacin) control groups. The repetitive amount used for each test was determined using the Federer formula. Based on the Federer analysis, 4 samples are required for each concentration test for the Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa experiments. Furthermore, to assess the Kirby Bauer antibacterial analysis test's strength, we performed a well diffusion antibacterial analysis test on Escherichia coli and Pseudomonas aeruginosa.

Disc Diffusion Method
The Kirby-Bauer method was performed using a modification of the disc diffusion method. Bacteria were grown on a solid medium. A 0.5 McFarland standard bacterial suspension was prepared in a sterile saline solution (NaCl 0.85% (w/v); Merck, Darmstadt, Germany) via overnight culturing in a flask containing Mueller-Hinton Broth. Respective bacterial suspensions were seeded on Mueller-Hinton agar (MHA; Merck, Germany) plates, and 6 mm sterile blank discs, which had already been soaked with respective concentrations of extract, were impregnated on top. The prepared plates were incubated at 37°C for 24 hours. The inhibitory zone was identified and recorded carefully (Hudzicki 2009).

Well Diffusion Method
Bacteria were grown on a solid medium. 0.5 McFarland standard bacterial suspensions (Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853) were prepared in sterile saline solution (NaCl 0.85% (w/v); Merck, Darmstadt, Germany) via overnight culturing in a flask containing Mueller-Hinton Broth. The well diffusion method involved making a 6 mm diameter hole in the MHA media containing the respective bacteria. The extract was then placed inside the hole carefully, and the prepared agars were incubated at 37°C for approximately 24 hours. The inhibitory zone was identified and recorded carefully (Balouiri et al. 2016).

Qualitative Phytochemistry
Phytochemical testing was carried out on extract and bio-oil. Phytochemical testing aims to determine the qualitative content of secondary metabolites contained in a sample.

Alkaloid Test
The extract or bio-oil was added 1 ml of 2N hydrochloric acid and 9 ml of water, heated in a water bath for 2 minutes, cooled, and filtered. Then, 3 ml of the filtrate was transferred to the watch glass, and two drops of Dragendorf reagent were added. If brown sediment occurred, the extract or bio-oil contained alkaloids. If a white or yellow clot residue is dissolved in methanol with the Mayer reagent, there is a positive for alkaloid substances.

Flavonoid Test
1 ml of the extract or bio-oil is evaporated, and the rest is dissolved in 1-2 ml of ethanol (95%) P. Then, 500 mg of zinc powder and 2 ml of 2N hydrochloric acid are added, the solution is allowed to stand for 1 minute, and then ten drops of concentrated hydrochloric acid are added. If the solution turned red in 2-5 minutes, it meant it contained flavonoids.

Tanin Test
Simplicia extract was added 50 ml of aquadest, boiled for 5 minutes, and then cooled. Then, 5 ml of the filtrate was transferred to a test tube, and FeCl 3 reagent was dropped in. If there was a greenishblack colour, that indicated the presence of tannin compounds.

Saponin Test
Extract or bio-oil was put into a test tube, 10 ml of hot water was added, and the mixture was cooled and then shaken vigorously for 10 seconds to form a stable white foam as high as 1-10 cm for not less than 10 minutes. If, with the addition of 1 drop of 2N hydrochloric acid, the foam did not disappear, that indicated that the extract or bio-oil contained saponins.

Steroid/Triterpenoid Test
The extract or bio-oil was added to 20 ml ether and macerated for 2 hours. Then, three drops of the filtrate were transferred to watch glass and dripped with a Lieberman-Burchard reagent (glacial acetic acid-concentrated sulfuric acid). If it turned red, that indicated the presence of steroid compounds, green indicated triterpenoid compounds.

Quantitative Phytochemistry
Quantitative Phytochemical testing was carried out on extract and bio-oil to measure the total alkaloids and polyphenols in the extract and bio-oil.

Total Flavonoid
A total of 10.0 mg of quercetin was put into a 10 ml volumetric flask and dissolved with ethanol until the limit mark to obtain a 1,000 µg/ml concentration. The standard solution of quercetin with a concentration of 1,000 µg/ml is used for the manufacture of series (3 µg/ml, 5 µg/ml, 7 µg/ml, 9 µg/ml, 11 µg/ml). Then from each concentration, 3 ml of ethanol, 0.2 ml of 10% AlCl 3 , 0.2 ml of 1M of Na acetate were added and distilled water to a volume of 10 ml. Then the standard solution at which concentration has been determined is left to stand for operating time, then the absorbance is read at a wavelength of 434.5 nm on a spectrophotometer.

Total Polyphenol
A total of 300 µl of a gallic acid solution with a 50 µg/ml concentration added with 1.5 ml of Folin-Ciocalteau reagent (1:10), then shaken and let stand for 3 minutes. The solution was added1 with 1.2 ml of 7.5% Na 2 CO 3 solution, shaken until homogeneous, and allowed to stand at room temperature in 0-90 minute. The absorbance was measured at a wavelength of 600-850 nm on a spectrophotometer.

Data Analysis
This experiment was performed in duplicate with a minimum of four repeats. Data were analyzed using a one-way parametric test with the statistical program SPSS 21.0 (Statistical Package for the Social Sciences). A parametric test can be done if the data variance is homogeneous and normal. An independent T-test was performed to equalize the bacterial tests with different concentrations and analyze the differences between the two extraction methods and two antibacterial activity assessments. The significance level for the difference was set at p<0.05.

Results
We have previously performed EHRE extraction methods (resulting extract) with ethanol solvent for Nicotiana tabacum Var Virginia leaves from Indonesia. This study performed the pyrolysis extraction method (resulting bio-oil) with propylene glycol solvent. The yields produced using the EHRE method are lower (23wt%) than those produced using pyrolysis extraction methods (47.6wt%). Higher nicotine yields of up to 31.1% were obtained using pyrolysis extraction compared to the EHRE method (6.3%). The EHRE method's extraction procedure was done in 6 hours with 1 atm and 150 rpm centrifuge speed (heated at a temperature of 80°C). In the pyrolysis method, the extraction is done in 50 minutes with 1 atm and heated at 500°C.

Qualitative Phytochemistry, Quantitative Phytochemistry, and Yield Extract Efficiency
Qualitative phytochemical tests are performed to determine the active compounds in tobacco leaf extracts, which can be useful as antibacterial agents. Based on the phytochemical test results of the tobacco leaf extracts examined in this study, it is confirmed that the active antimicrobial compounds in tobacco leaf extracts are alkaloids, flavonoids, saponin, tannin, phenolic, glicoside, and triterpenoid ( Table 1). The number of qualitative total alkaloids is greater in biooil, but total polyphenol was still higher in the tobacco extract (Table 2).
Although the polyphenol compound was found higher in the EHRE methods, on the contrary, nicotine yields showed the opposite around. The higher nicotine yields were obtained from pyrolysis extraction until 31.1% of yields (Table 3). The yields produced from 518 Pramono AP et al. The first analysis compared the antibacterial agents' effectiveness using different extraction methods, namely heat reflux (EHRE) and pyrolysis extraction, on several bacterial tests. The study results showed that the pyrolysis extraction method produced higher antibacterial potential than the heat reflux method (Figure 1). A significant statistically difference was only seen in Enterococcus faecalis, but not in other bacteria. Even so, pyrolysis is still better in inhibiting other bacteria even though it does not show significance in statistical tests.
The data also showed a significant difference in extracts' effectiveness ranging from 40 to 100% against Enterococcus faecalis compared to their effectiveness against the other three test bacteria (Figure 1). This study showed that Enterococcus faecalis is more susceptible to active antimicrobial substances in tobacco extracts and bio-oils. Enterococcus faecalis belongs to the susceptible bacteria in analyzing the potential antimicrobial differences between extractions methods, especially in EHRE and pyrolysis extraction methods.
When it comes to the well diffusion test, Nicotiana tabacum var Virginia's showed the highest antibacterial activity in bio-oil produced using the pyrolysis extraction method compared to the EHRE method, which is tested against Escherichia coli (Table 3). This is because of the pyrolysis extraction method that we have done has reached 47.6wt%, which is very much higher than the EHRE method, which is only 23wt%. An increase in extraction yield could be accompanied by an increase in Nicotiana tabacum var Virginia's antimicrobial compound and enhancement of the inhibition diameter. This is also supported by the GC-MS from our bio-oils results which show that dominant compounds in the biooils from the pyrolysis process were organic acids and pyridine (besides nicotine), which the antibacterial potential are well known. In contrast, the heat reflux extract was dominated by γ-sitosterol, where the antimicrobial potential is still unclear.
When comparing the extraction methods for the Nicotiana tabacum in this study with other previous extraction studies from the same plant species (Nicotiana tabacum sp.), it seems that the maceration method produces more significant inhibition than the pyrolysis method in this study (Table 4). The factor that influences these results must be studied further.
When comparing the antibacterial test, Nicotiana tabacum antimicrobial test against Escherichia coli with well diffusion in this study, with other previous studies against other bacteria, showed that E. coli has a higher susceptibility to the antibacterial compound in Nicotiana tabacum, compared to the different bacteria that have been examined (Table 4). It can reach up to 26.66 mm in inhibition diameter. This might be due to the initial concentration used for the experiment is quite high (3 g/ml) compared to the concentration used in the pyrolysis method (0.33 g/ml) in this study. Higher concentrations for increasing the diameter of inhibition in the pyrolysis method could be an alternative for further research. This is also seen in the antimicrobial test of other plant species against E. coli, where E. coli also show high susceptibility to active substances from other plant extracts as well.

Comparison of Disc Diffusion Antibacterial Test to the Well Diffusion Antibacterial Test against Escherichia coli and Pseudomonas aeruginosa
A second analysis was carried out to compare the differences in antibacterial activity for bio-oils used with different test methods, namely the disc diffusion test method and the well diffusion test method when deployed against the two test bacteria (Escherichia coli and Pseudomonas aeruginosa). The analysis results indicate that extracts implemented using the well diffusion test method create greater inhibition diameters with a significant difference (Figure 2).
Our study confirms previous studies' findings, which showed that the well diffusion method is more effective than the disc diffusion method. Is also confirm from previous studies against Escherichia coli using different plant extracts, except Adenium obesum, Syzygium aromaticum, and Capparis spinosa plant extracts produced using the maceration extraction method. In testing using Pseudomonas aeruginosa, it also showed a similar trend, except for Adenium obesum and Syzygium aromaticum plant extracts. 520 Pramono AP et al. Vol. 29 No. 4, July 2022

Pyrolisis Extraction Degraded Some Tobacco Active Compounds but Still Have Functional Bacterial Growth Inhibition
Qualitative total alkaloids are greater in biooil, but total polyphenol was still higher in the tobacco extract (Table 2). This study also shows that polyphenol compounds were less in bio-oil than tobacco extract (Table 2). This can be caused by the polyphenols themselves, which are easily degraded at high temperatures done in pyrolisis, compared to alkaloids (Hanuka Katz et al. 2020). Our study showed that the extraction of pyrolysis degraded some active tobacco compounds with antimicrobial potential. However, pyrolysis still showed higher antimicrobial activity than EHRE. The pyrolysis extraction can isolate more other antimicrobial components (such as nicotine) than EHRE (Table 3). However, also some components are degraded due to the use of hot temperatures. Previous research from Cardoso et al. 2017 on tobacco extraction using the pyrolysis method showed that the phenolic compounds found in the bio-oil were a less significant number than other compounds, such as nicotine, furfural, and acetic acid (Cardoso and Ataíde 2013).

Organic Acids and Pyridine (besides Nicotine) as Bio-oils Dominant Compounds (in Pyrolysis Extract) Showed Higher Antimicrobial Properties
Our study results showed that the pyrolysis extraction method produced higher antibacterial potential than the heat reflux method (Figure 1). This could be due to the higher number of alkaloids in pyrolysis. It may also be caused by differences in the dominance of the active substance produced by the heat reflux method and the pyrolysis in our study. Through the pyrolysis process, Bio-oil produces products with more varied compounds in higher concentrations and contain many active compounds. Our GC-MS analysis study showed that the bio-oils dominant compounds from the pyrolysis process were organic acids and pyridine, besides nicotine. Organic acid and pyridine have antimicrobial properties (Gozan et al. 2014). Unlike the tobacco extract produced by heat reflux, pyridine is not the dominant substance found other than nicotine, but rather γ-sitosterol (Andjani et al. 2019).
The pyridine ring is a compound with a spectrum of biological activity the antimicrobial potential. Pyridine has antimicrobial potential against several bacteria (Desai et al. 2017;Radwan et al. 2020).
Some plants that contain predominant γ-sitosterol have antimicrobial potential (Zhang et al. 2011;Silveira et al. 2017). The possibility from our results can be concluded indirectly that pyridine has more antimicrobial properties than gamma sitosterol. But how far gamma sitosterol can be antimicrobial compared to pyridine remains proven further.
Organic acids can inhibit bacterial growth by increasing the entry of transition metals into the bacteria. A strong synergy was found between low concentrations of non-toxic metals and organic acids, with an inhibitory effect on bacterial growth (Zhitnitsky et al. 2017). Previous studies have also shown that organic acids have an inhibitory effect on Gram+and Gram-bacteria (Kovanda et al. 2019).
In addition, the dominant antimicrobial action of pyrolysis can also be caused by the relatively higher number of alkaloids. The antibacterial mechanism of flavonoid compounds causes damage to the bacterial cell walls and disrupts cell metabolic processes (Cushnie and Lamb 2011;Khameneh et al. 2019). 522 Pramono AP et al.

Enterococcus faecalis is more susceptible to Active Antimicrobial Substances compared to other Bacteria for Both EHRE (extract) and Pyrolysis (Bio-oil) Extraction
Comparing the bacteria used in our test shows that Enterococcus faecalis are more susceptible to active microbial substances from Nicotiana tabacum var Virginia than the other three test bacteria (Figure 1) in both tobacco extracts and bio-oils. Some bacterial species are more susceptible to antimicrobial compounds, and some are innately resistant. This is due to each bacterial pathogen's unique profile characteristics and antibacterial compound mechanisms (Khameneh et al. 2019). This was also shown in previous studies (Chandrappa et al. 2015;Noushad et al. 2018).

Well Diffusion Test Method has a more Efficient Diffusion Process compared to the Disc Diffusion Method on Bio-oils against Escherichia coli and Pseudomonas aeruginosa
A second analysis of bio-oils on different test methods (disc diffusion test method and the well diffusion test method) against the two test bacteria (Escherichia coli and Pseudomonas aeruginosa) indicate that the well diffusion test method has greater inhibition diameters with a significant difference ( Figure 2). This may be due to the more efficient diffusion process of the well method. This result is confirmed by another study on antimicrobial tests (Valgas et al. 2007;Mansour Saleh Saif et al. 2017) and suggests that the well diffusion test method is more effective than the disc diffusion test method ( Figure  2). In another study on a different plant extract, the well diffusion test method produced a greater inhibitory zone diameter than the disc diffusion test method (Valgas et al. 2007;Mansour Saleh Saif et al. 2017). This may be because the tobacco leaf extract's osmolarity is higher in the well diffusion test method than in the disc diffusion test method. The well diffusion test method involves making a hole in the Muller Hinton Agar (MHA), filled with tobacco leaf extract. The tobacco leaf extract diffusion into the medium will be complete and homogeneous (Balouiri et al. 2016). In contrast to the well diffusion test method, in the disc diffusion test method, the tobacco leaf extract is not directly poured into the MHA; instead, the disc paper is first soaked in each component of each tobacco leaf extract before being placed on top of the MHA (Balouiri et al. 2016). In a well diffusion test against Escherichia coli from different plant extracts besides Nicotiana tabacum (Table 1), the disc diffusion test method produced a smaller inhibitory zone diameter when compared to the well diffusion test method because the greater absorption of the antimicrobial compound to the disc paper in the disc diffusion method made the process of antimicrobial compound diffusion to the media less than optimal (Valgas et al. 2007;Mansour Saleh Saif et al. 2017).
Moreover, another antibacterial test against Escherichia coli using different plant extracts showed the same trend, except the tests that used Adenium obesum, Syzygium aromaticum, and Capparis spinosa plant extracts using the maceration extraction method (Mansour Saleh Saif et al. 2017). Thus, it is most likely that the findings in our study on the well diffusion test method are more effective than the disc diffusion test method against Escherichia coli are comparable to the findings of other antibacterial test experiments (Elisha et al. 2017;Mansour Saleh Saif et al. 2017;Valgas et al. 2007).
Disc diffusion and well diffusion antibacterial testing with Nicotiana tabacum have been conducted against Pseudomonas aeruginosa, as shown in Table  1. Our study produced results in line with previous studies, which indicated that the well diffusion test method is more effective than the disc diffusion test method. Moreover, another antibacterial test against Pseudomonas aeruginosa using other plant extracts showed the same trend, except for where Adenium obesum and Syzygium aromaticum plant extracts derived using the maceration extraction method were used (Mansour Saleh Saif et al. 2017). Thus, it is most likely that the findings of this study that the well diffusion test method is more effective than the disc diffusion test method are comparable to the findings of other antibacterial test experiments against Pseudomonas aeruginosa. This could be due to the different antibacterial compounds that reside in plants. Some of these antibacterial compounds might be more effectively captured in the disc diffusion and inhibit bacteria growth more effectively. In contrast, other antibacterial compounds might work better in well diffusion test methods since they are abundant in liquid form in the well (Balouiri et al. 2016).
In conclusions, this study's antibiotic testing comparison indicates that bio-oils produced using the pyrolysis method are better than the extracts produced using the heat reflux method for extracting the antibacterial compounds from Nicotiana tabacum var. Virginia from Ponorogo, Indonesia. This is proven by our result, where the pyrolysis method produces a greater diameter of inhibition compared to the EHRE method, against Staphylococcus aureus (6.35 mm), Enterococcus faecalis (5.90 mm), Escherichia coli (3.97 mm), and Pseudomonas aeruginosa (5.025 mm). This is related to antimicrobial compounds inside in the bio-oils, where alkaloids are higher in bio-oils. However, fewer polyphenols are found in bio-oils, but there are still antimicrobial compounds predominantly found in bio-oils (pyridine) besides nicotine.
Moreover, our study showed that antibacterial using the well diffusion test method was more effective for delivering the antimicrobial compound than agar diffusion, indicated by the higher inhibition diameters produced against Escherichia coli. This research proves that bio-oil in the well diffusion test method showed the greatest antibacterial effectiveness. It created the biggest inhibition zone, with a maximum of 11.65 mm and 8.90 mm for E. coli and P. aeruginosa (Table 3). Nevertheless, the initial concentration used in the experiments, the test method and susceptibility of bacteria against the active antimicrobial compound were the factor that should be considered to influence the inhibition diameter enhancement on antibacterial testing.

Conflicts of Interest
The authors declare no conflict of interest. The founding sponsors had no role in the study's design; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.