Application of Sterilization Process for Inactivation of Bacillus Stearothermophilus in Biomedical Waste and Associated Greenhouse Gas Emissions

: This study investigated the biomedical waste collection, transportation, and treatment activities in the city of Kocaeli, Turkey. As an alternative to incineration technology, a steam autoclave was used to sterilize the biomedical waste. Information regarding the collection, transportation, treatment and associated greenhouse gas emissions (GHG) were also investigated. Prior to sterilization, biological indicator vials containing Bacillus stearothermophilus were placed in the center of the load to ensure that the pathogens were destroyed. GHG emissions were calculated based on the fuel consumed by the biomedical waste collection vehicles and the electricity / natural gas used at the sterilization plant. Results of this work revealed that the total biomedical waste generated per year increased from 1362 tons in 2009 to 2375 tons in 2019. The amount of biomedical waste generated per hospital bed was determined as 1.19 kg.bed − 1 .day − 1 . Results show that for e ﬃ cient sterilization of biomedical wastes, the steam treatment system process should be operated at a contact time of 45 min, a temperature of 150 ◦ C, and at a steam pressure of 5 bar. Biological indicator tests showed that the number of living Bacillus stearothermophilus decreased signiﬁcantly, with removal rates greater than 6log 10 . Finally, it was determined that the biomedical waste management activities generated a total of GHG emissions of 5573 ton CO 2 equivalency (tCO 2 -e) from 2009 to 2019. Furthermore, the average global warming factor (GWF) was calculated to be 0.269 tCO 2 -e per ton of biomedical waste generated. This study showed that the sterilization process is very e ﬀ ective in destroying the pathogens and the management of biomedical waste generates considerable amounts of GHG emissions.


Introduction
The appropriate management of biomedical waste is extremely important due to its significant environmental and health hazards. Recently, many attempts have been made to better manage the biomedical waste problem. Authorities define biomedical waste as the waste generated during the diagnosis and treatment of people and animals. If not properly handled, biomedical waste poses a great risk of infection through the spread of pathogens from health institutions into the environment [1]. Medical devices are now being manufactured for single use only, thus further increasing the amount of biomedical waste especially in developing countries. This will result in a rapid increase in biomedical waste amounts that should be disposed in a safe manner [2]. In the literature, there are different names for biomedical wastes such as hospital waste, regulated biomedical waste and infectious waste [3,4]. The terms infectious waste and biomedical waste are usually used for wastes that cannot be disposed of in a municipal solid waste landfill due to their pathogenic content. The safe disposal of biomedical wastes is of a great concern for the generators and the public. Different treatment methods can be applied in the treatment of biomedical waste. The main purpose in the treatment 2 of 14 of biomedical wastes is to make it safe for human and environmental health. The methods used to make biomedical waste harmless can be grouped as incineration, sterilization, plasma pyrolysis, and microwaving. In the US, about 60% of biomedical waste is incinerated, 37% is sterilized, and the rest is treated by different methods [4]. Within the scope of medical waste statistics, it was reported that 89,545 tons of medical waste was collected from 1550 health institutions operating as of the end of 2018 in Turkey. The amount of medical waste collected in 2018 increased by 4% compared to the previous year. Of this amount, 92.3% of the medical waste collected was sterilized and 7.7% was sent to incineration facilities [5]. Alternative treatment methods to incineration have always been the focus of biomedical waste generators. For example, sterilization or autoclave methods use shredders to reduce the waste volume. Sterilization inactivates microorganisms by using the saturated steam and is commonly used to treat infectious biomedical waste [6,7]. Thermal processes are applied as low, medium and high temperature depending on the process temperature applied. As a method, the thermal process is applied as the wet (steam) and dry heat treatment. In dry heat treatment, heat is applied to biomedical waste without adding water or steam. Heat is delivered to the waste by conduction, convection or thermal radiation. The processing time and the temperature to be applied depend on the characterization and quantity of the biomedical waste treated. The process temperature to be applied should not be too high to prevent the volatile organic compounds that can be released from the plastic wastes but should be sufficient for the sterilization of waste [6]. The process of sterilization is the treatment of biomedical wastes with steam at high temperature and pressure. If the temperature and contact time are sufficient, this process inactivates many types of microorganisms. Biomedical waste containers are placed in a closed chamber and sterilized with steam for a certain time at the required pressure and temperature. As a general practice, biomedical waste is steamed at 121 • C for 30 min at 2 bar and approximately 99.99 percent of microorganisms are inactivated by this process [8][9][10]. Biomedical wastes can be landfilled together with municipal solid wastes after steam treatment and size reduction.
Sterilization is the process of completely destroying all kinds of microbial life, including bacterial spores in biomedical wastes, by physical, chemical, and mechanical methods, or reducing the level of these microorganisms by 99.9999% (6 log 10 reduction). Whether the biomedical wastes treated by sterilization are rendered harmless is tested using chemical and biological indicators. Chemical indicators are used in the autoclave sterilization of biomedical waste. When the sterilization is completed, color change must be detected in the chemical indicator carrier that has been autoclaved together with the waste. In the biological indicator test, the viability of the biological indicator is used to detect whether all potential infectious microorganisms have been destroyed in the sterilized waste. It is a tubular test indicator with Bacillus stearothermophilus, which is known to be the most resistant microorganism to heat. If the test result is negative, the sterilized biomedical waste is sent to the landfill, but if the test result is positive, the sterilization process should be repeated. Ananta, Heinz [11] reported that Bacillus stearothermophilus spores can be inactivated by high-pressure treatment, but only if it is applied at an elevated temperatures. Rajan, Pandrangi [12] also reported that, while the thermal inactivation of spores followed first-order kinetics, the Weibull model best described the inactivation of Bacillus stearothermophilus spores. Iciek, Papiewska [13] conducted a study to investigate the combined effect of temperature, pH and NaCl concentration on the thermal inactivation of Bacillus stearothermophilus and observed that the sterilization temperature and pH of the sterilized medium as well as the concentration of NaCl, had a significant effect on spore activation and destruction.

Greenhouse Gas Emissions
Global greenhouse gas (GHG) emissions have grown since pre-industrial periods, with a 70% increase between 1970 and 2004 [14]. Since pre-industrial times, increased greenhouse gas emissions from human activities have caused a significant increase in the atmospheric greenhouse gas concentrations. Between 1970 and 2004, global emissions of CO 2 , CH 4 , N 2 O, hydrofluorocarbons (HFCs), perfluorocarbons (PFCs) and sulphur hexafluoride (SF6), described by their global warming potential (GWP), have increased by 70% from 28.7 to 49 Giga ton (Gt) CO 2 -eq. CO 2 emissions grew by approximately 80% between 1970 and 2004, representing 77% of the total greenhouse gas emissions in 2004. Between 1970 and 2004, the greatest growth in global greenhouse gas emissions came from the energy supply sector, with an increase of 145%. The Intergovernmental Panel on Climate Change (IPCC) predicts that global greenhouse gas emissions will continue to increase over the next few decades [14]. However, IPCC also estimates that studies have demonstrated a significant economic potential for reducing global greenhouse gas emissions over the next decades that could balance the projected growth of global emissions or reduce emissions below current levels. GHG emissions will then need to peak and decline to stabilize greenhouse gas concentrations in the atmosphere. The lower the level of stabilization, the faster this peak and drop will have to occur. Reduction efforts over the next two to thirty years will have a major impact on opportunities to reach lower levels of stability.
The generation, transportation and disposal practices of wastes potentially generate greenhouse gas emissions [15]. Total GHG emissions resulting from waste management activities in the world are about 1.3 GtCO 2 -e, corresponding to about 2.8 percent of total GHG emissions [14]. Approximately, 3.3% of total greenhouse gas emissions originate from waste management activities in Turkey [16]. The total greenhouse gas emissions of Kocaeli city for 2016 were calculated as 25.1 million tons of CO 2 -e. Of the total greenhouse gas emission, 65.3% of total emissions were from fixed sources, 17.4% from industrial processes, 15.0% from transportation, 1.4% from land use and 0.9% from waste management [17]. The collection, transportation and transfer of waste is not included in waste management activities, but in the estimation of mobile greenhouse gas resources (cars, trucks) [17]. The units of GHG emissions are converted into CO 2 equivalency (CO 2 -e) in order to better identify and evaluate GHG emissions. Another term commonly used to describe GHG emissions is called global warming factor (GWF). The GWF identifies the amount of GHG emissions generated per ton of biomedical waste collected, transported and sterilized. GWF used in this study is based on a 100-year time period as reported in the recent IPCC assessment report [18]. In the literature, there is no study that investigated the GHG emissions from biomedical waste collection and treatment systems in the city of Kocaeli, Turkey. This study had two main objectives. The first objective was to verify if efficient biomedical waste treatment can occur under standard operating parameters in steam treatment systems in the city of Kocaeli, Turkey. The second objective was to investigate the greenhouse gas (GHG) emissions generated during the transportation and treatment of biomedical waste.

Description of the Study Area
This study was conducted in the city of Kocaeli, which has a population of 1,875,493 and is located in the northwest of Turkey ( Figure 1). According to the Turkish Statistical Institute, 81,024 tons of biomedical waste were collected and treated by sterilization, incineration and other methods in 2016 in Turkey [5]. Based on this biomedical waste amount, about 450 million healthcare facility visits were recorded in 2016. Generally, biomedical wastes are segregated and placed in 10-L durable red plastic bags in the study area. Sharps and needles are first collected in yellow rigid plastic boxes and then placed in red plastic bags. As a safety rule, 1/3 of the capacity of the bags is always left empty. After tying the bags securely, they are temporarily stored in designated rooms and collected daily by licensed collection vehicles. Kocaeli Metropolitan Municipality has 9 biomedical waste collection vehicles operating for the 27 healthcare institutions and other small clinics. The collected biomedical waste is transported to an 8 ton.day −1 capacity sterilization plant located at the Kocaeli landfill site.

Collection and Transportation of Biomedical Waste
The biomedical waste management system implemented in Kocaeli city includes the disposal of all biomedical wastes originating from all public hospitals, private hospitals, dialysis centers, family health centers, laboratories and district municipalities within the borders of the municipality. Biomedical wastes, excluding pathological and hazardous wastes, generated in the boundaries of Kocaeli city and district municipalities are collected and sterilized at the biomedical waste sterilization facility within the scope of national biomedical waste regulation and then disposed in the solid waste landfill. Biomedical waste amounts from the public hospitals, private hospitals, dialysis centers, family health centers and laboratories in the study area are given in Table 1.

Collection and Transportation of Biomedical Waste
The biomedical waste management system implemented in Kocaeli city includes the disposal of all biomedical wastes originating from all public hospitals, private hospitals, dialysis centers, family health centers, laboratories and district municipalities within the borders of the municipality. Biomedical wastes, excluding pathological and hazardous wastes, generated in the boundaries of Kocaeli city and district municipalities are collected and sterilized at the biomedical waste sterilization facility within the scope of national biomedical waste regulation and then disposed in the solid waste landfill. Biomedical waste amounts from the public hospitals, private hospitals, dialysis centers, family health centers and laboratories in the study area are given in Table 1. In the study region, biomedical wastes are accumulated separately, where they occur, without being mixed with other wastes. Sharps, needles, infectious wastes, hazardous wastes and pathological wastes are collected in appropriate containers, which are compatible with the waste. The collected biomedical waste is first taken to the temporary biomedical waste storage of the health institution and then delivered to the biomedical waste collection vehicle. Once the biomedical waste is brought to the disposal site, the sharps, needles and infectious wastes are subjected to sterilization, while the pathological wastes and hazardous wastes are incinerated at the hazardous waste incineration plant located near the sterilization plant. The biomedical waste management system used in this study is given in Figure 2.
Appl. Sci. 2020, 10 In the study region, biomedical wastes are accumulated separately, where they occur, without being mixed with other wastes. Sharps, needles, infectious wastes, hazardous wastes and pathological wastes are collected in appropriate containers, which are compatible with the waste. The collected biomedical waste is first taken to the temporary biomedical waste storage of the health institution and then delivered to the biomedical waste collection vehicle. Once the biomedical waste is brought to the disposal site, the sharps, needles and infectious wastes are subjected to sterilization, while the pathological wastes and hazardous wastes are incinerated at the hazardous waste incineration plant located near the sterilization plant. The biomedical waste management system used in this study is given in Figure 2.

Sterilization of Biomedical Waste
In this study, a steam autoclave was used to sterilize the pathogens. Autoclaving is an efficient wet heat treatment and disinfection process. The steam autoclave was operated during this study at a contact time of 45 min, a temperature of 150 °C, and at a steam pressure of 5 bar. The minimum time required for contact depends on factors such as the temperature applied, the moisture content of the waste and the penetration of steam into the waste [9]. The following steps were applied during the biomedical waste treatment in this study. 1-Pre-heating: hot steam was injected into the jacket of the autoclave to pre-heat the autoclave; 2-Waste loading: waste containers were loaded into the autoclave. During the loading, the chemical and biological indicators were placed in the middle of

Sterilization of Biomedical Waste
In this study, a steam autoclave was used to sterilize the pathogens. Autoclaving is an efficient wet heat treatment and disinfection process. The steam autoclave was operated during this study at a contact time of 45 min, a temperature of 150 • C, and at a steam pressure of 5 bar. The minimum time required for contact depends on factors such as the temperature applied, the moisture content of the waste and the penetration of steam into the waste [9]. The following steps were applied during the biomedical waste treatment in this study. 1-Pre-heating: hot steam was injected into the jacket of the autoclave to pre-heat the autoclave; 2-Waste loading: waste containers were loaded into the autoclave. During the loading, the chemical and biological indicators were placed in the middle of the waste load to monitor the sterilization effectiveness. The autoclave door was closed and sealed; 3-Air discharge: air was discharged through pre-vacuuming; 4-Steam treatment: steam was injected into the autoclave chamber until reaching the required temperature; 5-Steam discharge: steam was discharged from the autoclave, by using a condenser; 6-Waste unloading: the treated waste was removed together with the chemical and biological indicator strips; and 7-Mechanical treatment: the treated waste was introduced into a shredder before the final disposal in the Kocaeli landfill.

Biological and Chemical Testing
The chemical indicator used at every charge was in the form of a strip and was removed from the autoclave tank together with the biomedical waste at the end of each treatment period. The chemical indicator was used if the tank had reached a sufficient temperature by changing the color on the strip. The biological indicator used in the control of the sterilization process was applied for one charge a day as stated in the Turkish biomedical waste regulation. According to the Turkish biomedical waste regulation, sportive bacteria Bacillus stearothermophilus or Bacillus subtilis standard origins should be used as biological indicators, because these microorganisms are more resistant to high humidity and high temperatures than other disease-causing microorganisms. A minimum reduction of 4 log 10 -6 log 10 is required in Bacillus stearothermophilus or Bacillus subtilis bacteria spores for the sterilization process to be considered valid. To control this, a certain number of Bacillus stearothermophilus spore-inoculated test strips were placed in the middle of the waste in a heat-resistant and vapor permeable container and the system was operated under normal conditions. At the end of the process, the test strips were removed from the waste. At the same time, at least one untreated biological indicator strip was also cultured in parallel as the positive control and incubated for 48 h at 30 • C for Bacillus stearothermophilus.
Since it is difficult to determine whether all microbial activities were completely destroyed, a probability function was defined at the end of sterilization based on the number of microorganisms that have survived. This function is often referred to as the reduction of the microorganisms that are most resistant to the sterilization process. Inactivation used today is defined as log 10 reduction. This is defined as the difference between the logarithmic numbers of test organisms that can survive before and after the sterilization process and can be expressed by the formula as follows: log 10 (cfu/g) R = log 10 (cfu/g) TO − log 10 (cfu/g) OS where log 10 (colony forming unit (cfu).g −1 ) R is the logarithmic number of reduction (R) of test organisms, log 10 (cfu.g −1 ) TO is the number of test organisms (TO) tested in the sterilization unit, log 10 (cfu.g −1 ) OS is the number of test organisms that survived (OS) after sterilization, and cfu.g −1 is the microorganism colony formation in 1 g of waste. At the end of the sterilization period, the tank was discharged and the sterilized wastes were loaded into the shredder. Then, the shredded wastes were disposed of in a municipal soil waste landfill located near the sterilization plant.

Greenhouse Gas Emissions
In order to determine the total diesel fuel used by the collection vehicles, an average fuel consumption of 0.5 L per km traveled was selected [20]. Fruergaard, Astrup [21] reported that for each 1 L of diesel fuel, 0.5 kg CO 2 -e was generated for provision and 2.7 kg CO 2 -e for combustion, which gives a total of 3.2 kg CO 2 -e.L −1 . These values were selected to calculate the total GHG emissions in this study. Upon determining the total amount of GHG emissions, a GWF for each year was estimated by dividing the total amount GHG emissions by the yearly total collected and treated biomedical waste. The amounts of GHG emissions resulting from electricity consumption at the sterilization plant was determined by using the emission factor of 0.480 kg CO 2 -e.kWh −1 provided for Turkey by the International Energy Agency (IEA) [22]. The amount of GHG emissions from natural gas consumption at the sterilization plant was determined by using the conversion factor of 10.34 kWh.m −3 natural gas (1000 Btu.ft −3 ) for electricity/natural gas energy equivalence [23].

Quantification of Biomedical Waste
Results of this study showed that the total biomedical waste generated per year increased from 1362 tons in 2009 to 2375 tons in 2019 ( Table 2). The amount of biomedical waste generated per hospital bed varied between 0.21 and 2.00 kg.bed −1 .day −1 with an average value of 1.19 kg.bed −1 .day −1 as of December 2019. This range seems to be similar compared to a study conducted in Istanbul, in which the daily averages of biomedical waste amount per hospital varied from 0.43 to 1.68 kg.bed −1 .day −1 [20]. The average diesel fuel consumed per kg of medical waste collected and transported was calculated as 0.041667 L kg −1 . In addition, the average electricity and natural gas consumed at the sterilization plant was calculated as 0.00944 kWh kg −1 and 0.02687 m 3 kg −1 , respectively.

Inactivation of Bacillus Stearothermophilus
In order to determine whether all the potentially infectious microorganisms were destroyed in the waste from the sterilization process, it needed to be checked whether the biological indicator microorganisms treated with the waste remained alive or dead. According to the Turkish biomedical waste regulation, a minimum reduction of between 4 log 10 and 6 log 10 is required in Bacillus stearothermophilus bacteria spores for the sterilization process to be considered valid. This was done by placing a certain number of Bacillus stearothermophilus spores containing an inoculated test indicator in the middle of the waste in a heat-resistant and vapor-permeable tube. At the end of the charge, the tube containing Bacillus stearothermophilus was taken from the waste, and the appropriate medium described by the producer of the biological indicator was plated on an agar medium. Meanwhile, at least one biological indicator that had not been subjected to sterilization was cultured as a positive control and incubated for 48 h at 55 • C for Bacillus stearothermophilus. In the chemical indicator test, when the result of the examination was negative, these biomedical wastes were re-sterilized by adding a biological indicator. These wastes were kept in biomedical waste temporary storage area until the biological indicator tests were completed. Even if there was no microbial reproduction as a result of the biological indicator, these wastes were re-sterilized. In this study, biological indicator tests showed that, with a contact time of 45 min, a temperature of 150 • C, and at a steam pressure of 5 bar, the number of living Bacillus stearothermophilus decreased significantly. Daily bioindicator tests showed that the removal rates for Bacillus stearothermophilus were always greater than 6 log 10 .

Greenhouse Gas Emissions
A total of 20,722,957 kg (≈20,723 tons) biomedical waste was generated in the study area between 2009 and 2019. It was confirmed by the authorities that each biomedical waste collection and transport vehicle carried approximately 1 ton of waste in each trip to the sterilization plant. Thus, the total trip numbers between 2009 and 2019 were 20,723, which included a round-trip drive from the first collection point to the sterilization plant and back to the same collection point. It was calculated, based on the information provided by the authorities, that approximately 863,457 L of gasoline was consumed by the biomedical waste collection vehicles between 2009 and 2019. For the calculation of fuel usage, an average diesel consumption of 0.5 L per 1 km traveled was selected, which was also recommended by Korkut [20]. Yearly average electricity and natural gas consumptions at the sterilization plant were provided by the plant operator as shown in Table 2. The total amounts of electricity and natural gas consumed between 2009 and 2019 were 195,616 kW and 556,068 m 3 , respectively. Table 3 summarizes the basic data and parameters used in the GHG calculation [21][22][23].   Figure 3 that the greater the amount of biomedical waste collected and sterilized, the more GHG emissions generated. As the amount of biomedical waste has increased over time, the amounts of GWF decreased from 0.272 to 0.265 tCO 2 -e.ton −1 . Figure 4 shows the amount of GHG emissions resulting from different fuels for biomedical waste collection, transport and treatment between 2009 and 2019. The amount of diesel fuel used by the biomedical waste collection and transport vehicles was used to calculate the GHG emissions from these activities. On the other hand, natural gas and electricity were only consumed at the sterilization plant for different purposes such as lighting, heating and running the autoclaves. The highest GHG emissions were observed from the natural gas use at the sterilization plant. Yearly GHG emissions from diesel combustion varied from 177 tCO 2 -e to 312 tCO 2 -e during the study period. However, GHG emissions from electricity consumption at the sterilization plant was much lower compared to that of diesel fuel combustion and natural gas use.  Figure 4 shows the amount of GHG emissions resulting from different fuels for biomedical waste collection, transport and treatment between 2009 and 2019. The amount of diesel fuel used by the biomedical waste collection and transport vehicles was used to calculate the GHG emissions from these activities. On the other hand, natural gas and electricity were only consumed at the sterilization plant for different purposes such as lighting, heating and running the autoclaves. The highest GHG emissions were observed from the natural gas use at the sterilization plant. Yearly GHG emissions from diesel combustion varied from 177 tCO2-e to 312 tCO2-e during the study period. However, GHG emissions from electricity consumption at the sterilization plant was much lower compared to that of diesel fuel combustion and natural gas use.    Figure 4 shows the amount of GHG emissions resulting from different fuels for biomedical waste collection, transport and treatment between 2009 and 2019. The amount of diesel fuel used by the biomedical waste collection and transport vehicles was used to calculate the GHG emissions from these activities. On the other hand, natural gas and electricity were only consumed at the sterilization plant for different purposes such as lighting, heating and running the autoclaves. The highest GHG emissions were observed from the natural gas use at the sterilization plant. Yearly GHG emissions from diesel combustion varied from 177 tCO2-e to 312 tCO2-e during the study period. However, GHG emissions from electricity consumption at the sterilization plant was much lower compared to that of diesel fuel combustion and natural gas use.

Discussion
In this study, the average amount of biomedical waste generated per hospital bed was calculated as 1.19 kg.bed −1 .day −1 . In one study, Mato and Kassenga [24] reported biomedical waste amounts varying between 0.84 and 5.8 kg.bed −1 .day −1 . Abu-Qudais [25] found at five Jordanian hospitals that the average daily biomedical waste generation rates varied between 0.29 and 1.36 kg.bed −1 .day −1 . In a similar study conducted at Italian hospitals, the biomedical waste generation rates varied between 0.2 and 3.5 kg.bed −1 .day −1 [26].
Daily routine bioindicator tests have shown that the removal rates for Bacillus stearothermophilus were always higher than 6 log 10 in this study. Sterilization effectiveness varies with many parameters that affect the heat transfer and vapor penetration, such as waste contents, waste density, moisture content, and container types [27,28]. In order to prevent any damage on the shredder, it was ensured in this study that the incoming waste was always free of metal objects. Sterilization efficiency is usually observed by giving a level of assurance at 10 −3 or 10 −6 , which indicates that there is a chance in thousands and millions, respectively. Essentially, this means that at least 3 or 6 log 10 pathogen reductions should be maintained. This level of reduction is usually possible because steam sterilization autoclaves are generally operated at minimum standards (121 • C for 30 min or 134 • C for 5 min) [8,10]. The steam autoclave in this study performed at a contact time of 45 min, a temperature of 150 • C, and at a steam pressure of 5 bar. However, some studies claim that these parameters are not sufficient for the complete sterilization of all biomedical waste types [28,29]. For instance, the inclusion of a grinding system before sterilization allows better sterilization due to a larger waste surface area for steam. Shredding transforms waste into an unrecognizable form and provides a volume reduction of up to 80% [7]. The shredder system achieved approximately 70 to 80% volume reduction throughout this study. It should be noted, however, that the use of integrated shredders and autoclaves can cause repeated failures and high maintenance costs [30][31][32]. As stated by the World Health Organization (WHO), in order to select the best biomedical waste treatment technology, they must pose minimal human health impact, minimal environmental impact, and must be cost-effective and easily implemented [33].
The advantages and disadvantages of five biomedical treatment technologies are summarized as follows [34]: 1-Landfilling: it is one of the oldest methods for biomedical waste disposal in undeveloped countries. However, biomedical waste landfilling includes some serious disadvantages such as the contamination of soil and water, the spreading of pathogens, and high GHG emissions. The only advantage of the landfilling of biomedical waste is the low cost and easy operation; 2-Sterilization: this process is preferred in several applications because it offers many advantages such as excellent efficiency, short treatment times, lower cost, minimum GHG emissions and air pollutants, environment-friendly technology, and the availability of wide range of autoclave sizes. However, sterilization has some disadvantages, such as the odor problem, unsuitability for hazardous and pathological wastes, and shredder requirement; 3-Incineration: the incineration of biomedical waste is suitable for all waste types, provides a high volume reduction, has a potential for energy recovery, and provides complete sterilization. The disadvantages of incineration include the following: a-the equipment is more costly to operate than the other alternatives, b-the process must meet the stringent regulatory requirements of air pollution control, c-heavy metals are usually found in the ash, d-it produces high GHG emissions, and e-dioxins and furans can be generated; 4-Microwaving: microwaving is another technology that can be used for biomedical waste treatment. The advantages of microwaving technology include the following, a-it is an environment-friendly technology, b-it offers high volume reduction, c-it produces no liquid waste, and d-it generates minimum air pollutants. The disadvantages of microwaving include; high cost, not suitable for all waste types, odor problems, and high GHG emissions; and 5-Plasma pyrolysis: this process has the following advantages, a-it is suitable for all types of wastes, b-it occupies less space, c-it is environmentally sound, d-it does not require a chimney, e-toxic residuals are minimum, f-it does not require segregation, g-energy recovery is possible, and h-it can reduce the waste volume by over 90%.
The collection and transport of biomedical waste would likely result in GHG emissions similar to the GHG emissions from municipal solid waste (MSW) collection and transport activities. However, treatment systems for MSW and biomedical waste are completely different, except for incineration. Therefore, GHG emissions from these different treatment systems for MSW and biomedical waste would also be different compared to each other. For instance, a net GWF of −0.274 tCO 2 -e.ton −1 was reported in the literature in a landfill gas (LFG) combustion unit, which indicated that 1 ton of MSW landfilled in order to generate electricity by burning LFG eliminated 0.274 tCO 2 of GHG emission [19]. In a similar LFG to energy study, Malakahmad, Abualqumboz [35] reported an average GHG emission of 0.291 tCO 2 -e.ton −1 . Yaman [19] reported GWF of −0.94 tCO 2 -e.ton −1 from the combustion of MSW (waste to energy), which indicated that the incineration process eliminates more GHG emissions than it generates. This of course takes into account the energy generated from the incineration process that would offset the additional GHG emissions arising from different energy generation systems. Similar to four different studies, GWFs of −0.01, −0.12, −0.2385, and −1.019 tCO 2 -e.ton −1 were also reported, respectively [36][37][38][39]. Khan, Khan [40] and Ali, Wang [41] conducted case studies and reported that the treatment and disposal of biomedical wastes can also be assessed according to their greenhouse gas emission rates.
The current waste management practices of countries can effectively reduce greenhouse gas emissions from the waste sector. For example, a wide variety of mature, environmentally friendly technologies are available to reduce emissions and provide common benefits for public health, environmental protection and sustainable development. Collectively, these technologies can directly reduce greenhouse gas emissions or prevent significant greenhouse gas generation. It also represents an important and growing potential for the indirect reduction of greenhouse gas emissions by minimizing, recycling and reusing waste, the conservation of raw materials, improved energy and resource efficiency, and the prevention of fossil fuel use. It should also be emphasized that there are high uncertainties regarding global waste greenhouse gas emissions resulting from national and regional differences in definitions, data collection and statistical analysis. Reducing greenhouse gas emissions from waste should be addressed in the context of integrated waste management. For instance, life cycle assessment (LCA) is an important tool to consider both the direct and indirect effects of waste management technologies and policies [42][43][44].

Conclusions
In this study, the collection, transport and steam sterilization of biomedical waste and the associated GHG emissions from these processes were investigated. The results of the steam sterilization system performed effective treatment for biomedical wastes containing needles, syringes and other non-hazardous and non-pathological infectious wastes. Bacillus stearothermophilus bacteria were investigated for the effectiveness of steam on bacteria during the sterilization process. It was observed during this study that the steam autoclave performed most effectively at a contact time of 45 min, a temperature of 150 • C, and at a steam pressure of 5 bar, to inactivate Bacillus stearothermophilus. Under these operational conditions, daily bioindicator tests showed that the removal rates for Bacillus stearothermophilus were always greater than 6log 10 .
It was shown in this study that the biomedical waste collection, transport and sterilization processes generated a total of GHG emissions of 5573 tCO 2 -e from 2009 to 2019. A large part of the GHG emissions generated in this study was from the combustion of diesel fuel by biomedical waste collection and transportation vehicles and the natural gas consumed at the sterilization plant. On the other hand, the use of electricity at the sterilization plant produced less GHG emissions than that of diesel and electricity use. Furthermore, the average GWF was calculated as 0.269 tCO 2 -e per ton of biomedical waste collected, transported and sterilized. The biomedical waste treatment by steam sterilization seems to be a safe and cost-effective treatment method compared to incineration, which can release hazardous air pollutants and GHGs. However, precautions should be taken to reduce the amount of GHG emissions by, for example, using electricity-powered biomedical waste collection vehicles, using solar energy panels on the roofs of sterilization plants, and also using biodegradable biomedical waste collection bags.
Life cycle assessment (LCA) can provide decision-support tools. There are many combined mitigation strategies that can be implemented cost effectively by the public or private sector, using LCA and other decision-support tools. Therefore, as a future work, a complete and comprehensive study of LCA should be conducted to determine the complete GHG emissions from medical waste collection, transport and sterilization process.