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

### **Cevat Yaman**

Environmental Engineering, College of Engineering, Imam Abdulrahman Bin Faisal University, Dammam 34212, Saudi Arabia; cyaman@iau.edu.sa

Received: 26 June 2020; Accepted: 21 July 2020; Published: 23 July 2020

**Abstract:** 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<sup>−</sup>1.day−1. Results show that for efficient 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 6log10. Finally, it was determined that the biomedical waste management activities generated a total of GHG emissions of 5573 ton CO2 equivalency (tCO2-e) from 2009 to 2019. Furthermore, the average global warming factor (GWF) was calculated to be 0.269 tCO2-e per ton of biomedical waste generated. This study showed that the sterilization process is very effective in destroying the pathogens and the management of biomedical waste generates considerable amounts of GHG emissions.

**Keywords:** infectious waste; sterilization; biomedical waste; greenhouse gas; *Bacillus stearothermophilus*

### **1. 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 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 [8–10]. 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 log10 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 CO2, CH4, N2O, 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) CO2-eq. CO2 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 GtCO2-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 CO2-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 CO2 equivalency (CO2-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.

### **2. Materials and Methods**

### *2.1. 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−<sup>1</sup> capacity sterilization plant located at the Kocaeli landfill site.

**Figure 1.** Location map of the study region [19].

### *2.2. 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.


**Table 1.** Amounts of biomedical waste generated in Kocaeli city in 2019.

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.

**Figure 2.** The biomedical waste management system, displaying the relevant steps.

### *2.3. 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.

### *2.4. 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 log10–6 log10 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 log10 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:

$$\log\_{10}\left(\text{cfu/g}\right)\text{R} = \log\_{10}\left(\text{cfu/g}\right)\text{TO} - \log\_{10}\left(\text{cfu/g}\right)\text{OS} \tag{1}$$

where log10 (colony forming unit (cfu).g−1) R is the logarithmic number of reduction (R) of test organisms, log10 (cfu.g<sup>−</sup>1) TO is the number of test organisms (TO) tested in the sterilization unit, log10 (cfu.g−1) OS is the number of test organisms that survived (OS) after sterilization, and cfu.g−<sup>1</sup> 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.

### *2.5. 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 CO2-e was generated for provision and 2.7 kg CO2-e for combustion, which gives a total of 3.2 kg CO2-e.L<sup>−</sup>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 CO2-e.kWh−<sup>1</sup> 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−<sup>3</sup> natural gas (1000 Btu.ft<sup>−</sup>3) for electricity/natural gas energy equivalence [23].

### **3. Results**

### *3.1. 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<sup>−</sup>1.day−<sup>1</sup> with an average value of 1.19 kg.bed<sup>−</sup>1.day−<sup>1</sup> 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<sup>−</sup>1.day−<sup>1</sup> [20]. The average diesel fuel consumed per kg of medical waste collected and transported was calculated as 0.041667 L kg<sup>−</sup>1. In addition, the average electricity and natural gas consumed at the sterilization plant was calculated as 0.00944 kWh kg−<sup>1</sup> and 0.02687 m<sup>3</sup> kg<sup>−</sup>1, respectively.

**Table 2.** Amounts of biomedical waste collected and treated from 2009 to 2019 and the associated fuel usages.


### *3.2. 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 log10 and 6 log10 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 log10.

### *3.3. 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 m3, respectively. Table 3 summarizes the basic data and parameters used in the GHG calculation [21–23].


**Table 3.** The basic data and parameters used in the global greenhouse gas (GHG) calculation.

Figure 3 shows the amount of GHG emissions and global warming factors (GWFs) generated during the transportation and treatment of biomedical waste from 2009 to 2019. The amounts of yearly GHG emissions from the consumption of diesel fuel were calculated based on the emission factor of 3.2 kg CO2-e.L−<sup>1</sup> [21]. The total amount of GHG emissions generated from the biomedical waste collection and transportation vehicles between 2009 and 2019 was calculated as 2763 tCO2-e.year−1. The International Energy Agency (IEA) reported that 1 kWh of electricity consumption in Turkey can generate 0.480 kg CO2-e [22]. Thus, by using the emission factor of 0.480 kg CO2-e.kWh−1, yearly GHG emissions from electricity consumption varied between 6.12 and 10.76 tCO2-e.year<sup>−</sup>1. The total amount of GHG emissions generated from the electricity usage at the sterilization plant between 2009 and 2019 was calculated as 93.9 tCO2-e.year−1. The conversion factor for the electricity/natural gas energy equivalence was taken as 10.34 kWh.m−<sup>3</sup> natural gas (1000 Btu.ft−3) [23]. Thus, by using the emission factor of 0.480 kg CO2-e.kWh<sup>−</sup><sup>1</sup> in this study, yearly GHG emissions from natural gas usage varied between 183.16 and 322.41 tCO2-e.year<sup>−</sup>1. The total amount of GHG emissions generated from the natural gas usage at the sterilization plant between 2009 and 2019 was 2758 tCO2-e.year<sup>−</sup>1. Finally, the total amount of GHG emissions resulting from the use of diesel fuel for waste collection and transportation vehicles, and electricity and natural gas uses at the sterilization plant, was calculated as 5573 tCO2-e.year<sup>−</sup><sup>1</sup> from 2009 to 2019 (Figure 3). The GWF values varied between 0.265 and 0.272 tCO2-e.ton<sup>−</sup>1, with an average value of 0.269 tCO2-e.ton−<sup>1</sup> of biomedical waste collected, transported and sterilized. It can be concluded from 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 tCO2-e.ton<sup>−</sup>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 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 3.** The amounts of greenhouse gases (GHG) versus the global warming factor (GWF) generated during the transportation and treatment of biomedical waste from 2009 to 2019.

**Figure 4.** Amounts of GHG emissions from different fuels for biomedical waste collection, transport and treatment between 2009 and 2019.

### **4. 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<sup>−</sup>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<sup>−</sup>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<sup>−</sup>1.day−<sup>1</sup> [26].

Daily routine bioindicator tests have shown that the removal rates for *Bacillus stearothermophilus* were always higher than 6 log10 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−<sup>3</sup> or 10<sup>−</sup>6, which indicates that there is a chance in thousands and millions, respectively. Essentially, this means that at least 3 or 6 log10 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 [30–32]. 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 <sup>−</sup>0.274 tCO2-e.ton<sup>−</sup><sup>1</sup> 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 tCO2 of GHG emission [19]. In a similar LFG to energy study, Malakahmad, Abualqumboz [35] reported an average GHG emission

of 0.291 tCO2-e.ton<sup>−</sup>1. Yaman [19] reported GWF of <sup>−</sup>0.94 tCO2-e.ton<sup>−</sup><sup>1</sup> 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 <sup>−</sup>0.01, <sup>−</sup>0.12, <sup>−</sup>0.2385, and <sup>−</sup>1.019 tCO2-e.ton<sup>−</sup><sup>1</sup> were also reported, respectively [36–39]. 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 [42–44].

### **5. 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 6log10.

It was shown in this study that the biomedical waste collection, transport and sterilization processes generated a total of GHG emissions of 5573 tCO2-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 tCO2-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.

**Funding:** This research received no external funding.

**Acknowledgments:** The author would like to thank Kocaeli Metropolitan Municipality (Turkey), IZMIT Waste and Residue Treatment Incineration and Utilization Corporation (IZAYDAS) for providing the data, and Imam Abdulrahman Bin Faisal University for academic support.

**Conflicts of Interest:** The author declares no conflict of interest.

### **References**


© 2020 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Review* **Trends in Biodiesel Production from Animal Fat Waste**

### **Fidel Toldrá-Reig 1,**†**, Leticia Mora <sup>2</sup> and Fidel Toldrá 2,\***


Received: 20 April 2020; Accepted: 21 May 2020; Published: 25 May 2020

**Abstract:** The agro-food industry generates large amounts of waste that contribute to environmental contamination. Animal fat waste constitutes some of the most relevant waste and the treatment of such waste is quite costly because environmental regulations are quite strict. Part of such costs might be reduced through the generation of bioenergy. Biodiesel constitutes a valid renewable source of energy because it is biodegradable, non-toxic and has a good combustion emission profile and can be blended up to 20% with fossil diesel for its use in many countries. Furthermore, up to 70% of the total cost of biodiesel majorly depends on the cost of the raw materials used, which can be reduced using animal fat waste because they are cheaper than vegetable oil waste. In fact, 6% of total feedstock corresponded to animal fat in 2019. Transesterification with alkaline catalysis is still preferred at industrial plants producing biodiesel. Recent developments in heterogeneous catalysts that can be easily recovered, regenerated and reused, as well as immobilized lipases with increased stability and resistance to alcohol denaturation, are promising for future industrial use. This manuscript reviews the available processes and recent advances for biodiesel generation from animal fat waste.

**Keywords:** biodiesel; fuel; energy generation; agricultural waste; food waste; animal waste; lard; tallow; animal fat; transesterification

### **1. Introduction**

Animal byproduct production, as part of the meat and poultry processing chain, is huge. For instance, it represents nearly 17 million tons per year only in the European Union [1]. Most of the waste results from over 328 million pigs, sheep, beef, goats and dairy cattle and 6 billion chickens, turkeys and other poultry that are slaughtered every year in Europe [2]. After rendering, materials classified as edible which amount up to 12 million tons, are processed in a variety of food and feed related sectors [3]. The remaining byproducts that are considered inedible have other applications for disposal such as biofuels and biodiesel for energy generation [4,5]. It is energy generation, especially biodiesel production, that is one of the most attractive and expanding applications [6]. In this sense, the production of biodiesel guarantees a better profitability of inedible animal byproducts. Animal waste also consists of the organic matter resulting from the meat processing industry as well as from human consumption. Biodiesel consists of mono-alkyl esters of long chain fatty acids produced from oil or fat, but the use of vegetable oil adds a high price to biodiesel, and this has prompted the use of animal fats as an interesting alternative. In addition to its renewability, biodiesel also constitutes an attractive alternative because it offers better lubricating properties than fossil diesel fuel and it is biodegradable and non-toxic. It also has an improved cetane number and high flash point [7]. Biodiesel also contributes to sustainability by reducing the carbon footprint due to lower CO2 emission compared to fossil diesel fuel [8]. CO2 is one of the most relevant gases because it contributes up

to 72% of greenhouse gases [9]. Biodiesel from animal fat achieves nearly 80% fossil CO2 reduction in comparison to 30% for soya [5]. In addition, the emission of polycyclic aromatic hydrocarbons is 75–90% lower than in conventional diesel, whereas total unburned hydrocarbon is 90% lower [10,11]. The emissions of sulfur and CO are also reduced [12].

The International Energy Agency reported that bioenergy must increase up to 25% until 2025 and continue to grow, and is estimated to reach 30% of the world's road transport fuel mix by 2050 [13,14]. Biodiesel is mostly produced from vegetable oils and animal fats and has been the object of research and development in recent decades. Research is directed towards the use of low grade feedstock, with the possibility of reusing catalysts and improving efficiency of reactors for transesterification [15]. In fact, oil and fat materials used as raw materials are of high relevance because they are estimated to represent 60–80% of the total cost of biodiesel production [16]. Therefore, it is important to select the best materials in each case because they are affected by the geographic location, climate and agriculture [11]. Several countries such as Malaysia, Indonesia, Argentina, USA, Brazil, and the Philippines, as well as countries in the EU, are using biodiesel as a good source of renewable and biodegradable energy [17]. A consumption of 17.4 million liters of biodiesel was reported in 2019 with 63% of this consumption by France, Germany, Spain, Sweden and Italy [18]. The total biodiesel world production in 2019 was approximately 35 to 45 million tons and is steadily increasing year by year [19]. The European Union is the world's largest biodiesel producer and, on an energy basis, represents nearly 75% of the total transport biofuels market. In fact, the European biodiesel industry has more than 202 plants and production in 2019 exceeded 14 million tons of biodiesel [18,20]. US production of biodiesel was more than 5.6 million tons in 2019 and came from 91 plants with a capacity of 8.3 million tons per year [21,22]. Biodiesel is usually blended up to 20% with fossil diesel fuels in most countries due to its complete miscibility and the unnecessary need for engine modification at such percentage. For instance, a 20% blend is used in the United States while at least a 10% blend is used in China [23]. Blends receive the name B5, B10 or B20 when the biodiesel volume content is 5%, 10% and 20%, respectively. Today, more than 78% of diesel vehicles coming off production lines are approved for up to B20 use [21]. For biodiesel to be blended with normal fossil diesel, it must comply in Europe with EN14214 from the European Committee of Standardization (ISO) and in the US with ASTM6751 from the American Society for Testing and Materials [5].

Biodiesel can be used in existing diesel engines without the need for substantial modification. Biodiesel has a higher oxygen content than conventional diesel and the carbon to hydrogen ratio is also lower. This explains the major advantages of biodiesel such as lower emission of particulate matter, but also a lesser content of sulfur, hydrocarbon and carbon monoxide [24,25]. The major challenge nowadays is the production of environmentally and economically viable biodiesel [23] and the use of animal fat waste could contribute towards achieving this goal. This review is highlighting the latest advances in the available processes for biodiesel production from animal fat waste.

To elaborate on the present review, the literature search was performed in Web of Science (WoS) database from 1 January 2010 to 28 February 2020. The terms "biodiesel production," "animal fat" and "transesterification" were used for the survey of published papers. In total, 1602 publications were found, 1594 of them in English, with most found over the last three years. Of these, 1541 manuscripts were research articles, 141 were reviews, 210 corresponded to meetings, 27 corresponded to books, and the rest fell under another category. Eligibility criteria were established as: (a) full-text with English language article; (b) articles selected were published in scientific journals that apply a peer review system; (c) books, workshops, conference reports, theses and case reports were excluded due to lack of peer review; (d) articles with other feedstocks like vegetable oils; and (e) articles that only analyzed the overall performance of the process and did not provide specific parameters for comparison or evaluation were not included. Titles and abstracts of manuscripts were further evaluated for selection of manuscripts and those selected were used for the current review. Some relevant publications published before 2010 were also considered as well as websites of producers and international

agencies with current data and updated information on industrial use of feedstocks, animal fats and biodiesel production.

### **2. Animal Fats as Feedstock**

About 328 million animals (cattle, sheep, pigs and goats) and 6 billion poultry (mainly chickens and turkeys) were slaughtered in 2014 in the European Union [2]. Such a high number of slaughtered animals produces enormous amounts of waste animal residue including fats that need to be treated to reduce pollution or recycled to give them some added value [26,27]. Such fats include beef tallow extracted from rendering fatty tissue of cattle, mutton tallow from rendering sheep, pork lard from rendering pigs and chicken fat from rendering feathers, blood, skin, offal and trims [28,29]. The wet rendering process includes the presence of water and fats heated below 49 ◦C. The goal is separation of the fat from the protein fraction. Other fats are those resulting from meat and the meat processing industry and those from recycling of the industrial cooking business. Such recycled greases that are produced from heated animal fats collected from commercial and industrial cooking can be classified as yellow grease and brown grease depending on the content of free fatty acids (FFA). Yellow grease is considered if FFA < 15% by weight and brown grease when FFA > 15% [30].

Typical fatty acid composition of pork lard, beef tallow, mutton tallow and poultry fats are shown in Table 1. There is a wide variety depending on the animal species [31] but in general, they contain common types of fatty acids, most of them having 16 to 18 carbons. Most relevant fatty acids are palmitic (16:0) and stearic (18:0) acids as saturated fatty acids (SFA); oleic acid (18:1) as monounsaturated fatty acids (MUFA); and linoleic (18:2) and arachidonic (20:4) acids as polyunsaturated fatty acids (PUFA) [32]. Due to their high content of saturated fatty acids (near 40% SFA), ruminant and pig fats are predominantly solid while those from chicken fat (nearly 30–33% of saturated fatty acids) are almost liquid or in semi-solid form [33]. Therefore, it must be taken into account that raw animal fats are mostly in a solid state at ambient temperature and therefore, preheating at 45 ◦C is required for their use as fuel for diesel engine [14]. Further, such high content in saturated fatty acids generally results in more stable biodiesel with high cetane numbers (more than 50 for lard and tallow).

One of the main applications of inedible animal fat byproducts is the production of biodiesel [6,34]. Inedible animal byproducts are structured into three categories within the EU that are defined according to their risk to human or animal health. Category 1 has the highest risk, Category 2 still offers a high risk, while Category 3 offers the lowest risk and is fit for human consumption although generally not used for human food because of its non-edible content or for commercial reasons. Major uses for Category 3 byproducts are as feed and pet food. In any case, fats from all three categories can be destined to biodiesel production and some stakeholders have reported that Category 3 provides better quality for biodiesel production [35]. Nearly 81% of caul and lung fat and 26% of cod and kidney, knob and channel fat from cattle are destined for biodiesel production. In the case of sheep fats, 88% of caul fat, 43.3% of lung fat and 67.1% of knob and channel fat are destined for biodiesel production [36]. In 2019, the total amount of vegetable oil and animal fat used as feedstock for biodiesel exceeded 13 million tons in the EU. From them, 800 thousand tons (6% of total feedstock) corresponded to animal fats, and such amount has remained fairly constant since 2014 [18,19]. In the case of the US, animal fats represented 8.4% of total feedstock and were poultry fat, tallow and white grease with amounts of 74, 132 and 243 thousand tons, respectively [21].


**Table 1.** Typical composition in major fatty acids (as % of total fatty acids) of pork, beef, mutton and chicken fats.

### **3. Transesterification for Producing Biodiesel from Animal Fats**

The major steps in the production of biodiesel from animal fat waste are shown in Figure 1. A pretreatment is needed because feedstocks like animal fats usually contain a high amount of free fatty acids (FFA) and water which reduces the yield of biodiesel [41] and increases production costs because of the difficulty of separation and purification [42,43]. Biodiesel is produced through the transesterification reaction of a fat with a short-chain alcohol in the presence of a catalyst. Different catalysts are available to be used for biodiesel production. Those most typically used in transesterification reactions are alkalis (sodium hydroxide, sodium methoxide, potassium hydroxide, potassium methoxide, sodium amide, sodium hydride, potassium amide and potassium hydride), acids (sulfuric acid, phosphoric acid, hydrochloric acid or organic sulfonic acid), heterogeneous catalysts like enzymes (lipases) and complex catalysts like silicates, zirconias, nanocatalysts, etc. [44]. A faster reaction rate of animal fats transesterification is obtained using alkali catalysts in comparison to acid catalysts [45,46] which are 4000 times faster [47] as well as less expensive and readily available [48]. Sodium and potassium hydroxides run quite well, and methoxides perform better but are more expensive [47]. Polyol-derived alkoxide base catalysts were prepared by heating aqueous sodium hydroxide solution and polyols under vacuum pressure [49] and potassium glyceroxide catalysts were produced from potassium hydroxide and non-volatile, non-toxic polyols like byproduct glycerol by heating and drying, making it cheap to produce. Furthermore, the rate of transesterification reactions in methanol was reported to be comparable to those observed for the conventional potassium methoxide catalyst under the same reaction conditions [50]. On the other hand, the use of acid catalysts for a transesterification reaction results in slower reaction rates and requires a higher alcohol to fat molar ratio and a larger reactor that may be subject to corrosion [51]. Acid catalysts are mainly used for reducing the free fatty acids content before its transesterification with alkaline catalysts [47].

The most commercially used short-chain alcohol for the transesterification reaction is methanol because of its cheap price, but other alcohols such as ethanol, propanol and butanol may also be used [52]. Transesterification consists of the conversion of triacylglycerols to diacylglycerols, releasing one fatty acid. Then, diacylglycerols are converted to monoacylglycerols, releasing a second fatty acid and, finally, monoacylglycerols are converted to glycerol, releasing a third fatty acid. In general, transesterification has a high conversion efficiency and low cost [53]. However, once a pretreatment has been performed [54], the efficiency of the transesterification reaction depends on many variables like the time and temperature of the reaction, type and molar ratio of alcohol, type and amount of catalyst used, the amount of water present in the reaction media, the composition of fatty acids and the release of free fatty acids to the reaction media. In industrial processing plants, approximately 100 kg of fat react with 10 kg of a short-chain alcohol (usually methanol) in the presence of an alkaline

catalyst, either sodium hydroxide or potassium hydroxide, to generate 100 kg of biodiesel and 10 kg of glycerin [55].

Examples of operating conditions during the transesterification step for production of biodiesel from animal fats are shown in Table 2. The optimum alcohol to oil molar ratio to get a biodiesel yield higher than 90% in alkaline catalyzed transesterification is reported to be around 6:1 which gives enough alcohol the capability to break the fatty acid–glycerol linkages. The use of a ratio greater than 6:1 gives a better yield in some cases but could hinder the glycerol separation process [56].

**Figure 1.** Major steps in the production of biodiesel from animal fat waste. Adapted from [46,47,53].


**Table 2.** Examples of operating conditions during the transesterification step for the production of biodiesel from animal fat waste.


**Table 2.** *Cont.*

As previously mentioned, if an alkaline catalyst is used for transesterification and there is a high content of free fatty acids, the reaction efficiency is drastically reduced because of the reaction of such free fatty acids with the catalyst resulting in soap formation [80,81]. This causes a loss of catalyst and ester product [82] and reduces the biodiesel yield to low levels, and therefore, the production costs increase [83]. The soap formation also makes glycerol separation and biodiesel purification difficult, which increases the cost of the resulting alkaline wastewater treatment [84]. The quality can also be affected due to side reactions producing unwanted products [85]. The free fatty acid content in animal fats is within the range of 5–30%, making a pretreatment necessary [26]. For an effective transesterification reaction, it is recommended that a limit in free fatty acids be equivalent to 1.0–1.5% [86], or an acid value below 2 mg KOH/g of oil [87]. There are different strategies in order to get such reduction in free fatty acids. For instance, waste pork fats containing free fatty acids within the range 4.9–13.5% were esterified for 4 h at 65 ◦C with 0.5 wt.% H2SO4 or 1.0 wt.% p-toluene sulfonic acid, keeping a 6:1 methanol to fat molar ratio, and the acid value was reduced below 2 mg KOH/g [61]. Furthermore, the waste fat of rendered pork was reported to have a high acid value of 4.3 mg KOH/g, but it could be reduced down to 0.75 mg KOH/g through a standard titration pretreatment method using sulfuric acid, even though it can be corrosive [23]. A continuous esterification process that reduced the reactor cost and reaction time, was designed for pretreatment deacidification using an ion exchange resin reaching an acid value reduced to 0.89 mg KOH/g and a conversion rate as high as 99.26% [80].

Thus, pretreatments are necessary to remove the excess of water, free fatty acids and suspended solids of animal fats before transesterification. Some of such pretreatments for moisture reduction are heat drying, silica gel, calcium chloride or anhydrous sodium sulfate [88–90]. The excess of free fatty acids may be removed by neutralization and separation [78]. Finally, the suspended matter may be removed by filtration under vacuum or through cellulose filters [88,90]. However, although the quality and yield of biodiesel are better, pretreatment steps result in additional costs [91].

An alternative process would be a two-step transesterification being the first step—an acid-catalyzed pretreatment to esterify the free fatty acids and thus, reducing their content—and the second step, where triacylglycerols undergo transesterification with the alkaline catalyst [52]. Other alternatives to consider are heterogeneous acid and base catalysts that have better tolerance to high water content and free fatty acids and can be re-used [82].

Heterogeneous catalysts offer the possibility of recycling and reducing the biodiesel production steps. Basic zeolites, alkaline earth metal oxides and hydrotalcites constitute some of the most used in recent literature. CaO is one of the most used catalysts and it has been loaded onto zeolite that offers a large surface area [15]. However, CaO can be poisoned when contacting water and carbon dioxide, making it not so attractive for industrial operation [92]. This catalyst could be recycled but the biodiesel yield is rapidly decreased down to 37.5% after its third use. Several nanocatalysts like nano CaO [48], activated carbon/CuFe2O4 encapsulated with CaO [69] and KF/CaO nanocatalysts [93] have been reported at the laboratory scale. Such nanocatalysts show a higher catalytic efficiency, better rigidity, larger specific surface area and better resistance to saponification [69].

Heterogeneous solid acid catalysts are reported to give lower yields. However, they have some advantages in the case of animal fats because they are insensitive to the problem of contents in free fatty acids and can perform esterification and transesterification simultaneously. Furthermore, the purification of biodiesel is avoided, and another advantage is the easier separation of the catalyst from the reaction products [94].

In cases where there is a high content of moisture and free fatty acids (e.g., animal fats), the enzymatically catalyzed transesterification is more attractive. Lipase converts both free fatty acids and triglycerides into biodiesel in the presence of an acyl acceptor. The main advantages are the mild reaction conditions, high selectivity and specificity of transesterification, broader substrate range, no soap formation, lower alcohol to oil ratio, lower requirements for purification and higher yields [95]. However, the major cost is due to the enzyme itself and its poor stability and longer reaction time, and the slow conversion rate because of the diffusion caused by its byproduct [96]. These troubles can be partly overcome through enzyme immobilization on an inert support that increases the enzyme stability, avoids the need for enzyme separation and improves process efficiency [97,98]. Other costs are derived from the deactivation of the enzyme at high molar ratios of alcohol to fat [96]. Three solutions have been proposed, which are using methanol stepwise addition, replacing methanol with an acyl acceptor like methyl acetate or ethyl acetate or using a solvent like t-butanol to get an improved solubility of methanol [99]. A variety of lipases from diverse microorganisms such as *Candida antarctica*, *Candida rugosa*, *Pseudomonas cepacia*, *Pseudomonas* spp. and *Rhizomucor miehei*, and immobilized lipases (i.e., Lipozyme® RMIM from *Rhizomucor miehei* or Novozym® 435 from *Candida antarctica*), have been reported in the literature [100]. It must be pointed out that transesterification with alkaline catalysis is still preferred at industrial plants producing biodiesel. In spite of much research on heterogeneous and enzyme catalysts, biodiesel producers have not yet adopted these technologies [101].

### **4. Quality of Biodiesel Produced from Animal Fat Waste**

Biodiesel has to comply with regulations EN14214 from the European Committee of Standardization (ISO) and the ASTM6751-3 from the American Society for Testing and Materials (ASTM). There are relevant benefits in the use of biodiesel produced from animal fats, such as the emission of polycyclic aromatic hydrocarbons being reduced by 75–90% and total unburned hydrocarbon by 90% when using biodiesel produced from animal fats when compared to conventional diesel [10,11]. The emissions of sulfur dioxide and CO are also reduced [12] as well as the particulate matter and nitrous oxides at part loads [18,66]. The properties of biodiesel produced from animal fats are compiled in Table 3. The cetane number reflects the ignition characteristics of the fuel. A high number is associated with better ignition quality [47]. The cetane number of biodiesels produced from animal fats is >50 and higher than that produced from vegetable oils due to their content of saturated fats (higher than 40%) and lower content of carbon and higher content of oxygen in comparison to conventional diesel [14]. The acid value is associated with the content in free fatty acids which, at high concentrations, can cause corrosion of the fuel supply system of the engine [66]. The cold filter plugging point (CFPP) represents the lowest temperature that a volume of liquid fuel will still flow through a given filter in a determined period of time when the fuel components start to gel or crystalize [102]. It is especially important for cold weather conditions. CFPP of biodiesel from animal fats is higher than 2 ◦C due to a higher content of saturated fatty acids. Flash point is the temperature at which the biodiesel exposed to a flame will ignite. Biodiesel from animal fats has a flash point higher than 150 ◦C and this provides better safety during transport and storage [47]. Biodiesel provides good lubricity that helps for a longer engine life [103]. However, the viscosity is higher than 3.5 mm2/s due to the saturated fats that give higher amounts of CH2 in the fatty acid methyl esters [104] which might create problems in pumping and combustion [11]. Finally, the percentage of free glycerin reflects the amount of glycerol remaining in the final biodiesel and, if the content is high, it could result in coking of the injector and damage to the fuel injection [47].

The cost of the obtained biodiesel from animal fat waste depends on several parameters such as the cost of the feedstock, the amount of free fatty acids and the type of necessary pretreatment, the type of catalyst, the operational maintenance and the type of purification for the biodiesel [105]. The refining of crude biodiesel obtained from animal waste is more costly than that from vegetable oils. Large amounts of glycerine (glycerol) are produced during transesterification which need to be removed because they influence the quality of the fuel and reduce the engine durability. The purification process (see Figure 1) usually consists of either wet washing based on water, dry washing based on adsorption and ion exchange, or novel methods based on liquid–liquid extraction, deep eutectic solvents or membranes. The purification step will thus remove glycerol as well as other impurities like residual catalyst, unconverted fats, and soap, and provides a better quality of biodiesel fuel [106]. About 1 kg of glycerol is produced for each 10 kg of biodiesel [107]. The recovered glycerol may be used for pharmaceutical, food, personal care biopolymers, or fuel additive applications although the value is low due to the large worldwide production of glycerol [92,105].


**Table 3.** Properties of biodiesel produced from animal fat waste.

### **5. Developments in Improving Biodiesel Production from Animal Fats**

China has been the most active country in publishing patents in the period 1999–2018 with 647 patents on biodiesel. The US had 266 patents, with more than 50% of them focused on reactors technology and processing methods [11]. There has also been patenting activity on pretreatment methods as well as on catalysts for improving the transesterification process. Specific examples of patents for biodiesel production from animal fat waste are shown in Table 4.



Technologies for process intensification like ultrasonic and microwave have been developed to be applied in transesterification and improve biodiesel production. The goal is to improve the miscibility of oils and methanol and thus increase the yield of the transesterification [73,117]. Immiscible liquids can be emulsified at an industrial scale through the use of low frequency ultrasonic irradiation. In the case of microwave irradiation, reactants can be efficiently and rapidly heated to the target temperature [118]. Other process intensification technologies like static mixers [119], capillary reactors [120,121], microreactors [122,123] or oscillatory flow reactors [124] are also intended to accelerate the reaction rate and enhance biodiesel production.

The use of microwave heating for animal fats containing up to 20% free fatty acids allowed for a decrease in the required time for free fatty acid reduction and increased the final yield [66]. Another alternative was the use of supercritical methanol with temperatures of 300 ◦C–400 ◦C, pressures up to 41.1 MPa, alcohol to fat ratios of 3:1 and 6:1, and short time (between 2–6 min) that gave 88% conversion for chicken fat [125]. The yield of biodiesel obtained with refined lard could also be obtained with waste lard containing fatty acids and water with no need for pretreatment [72]. Supercritical processes give faster reaction rates with no catalyst and avoid the need for pretreatment even in the presence of free fatty acids and water associated with the use of animal fats [30]. Other authors have assayed the transesterification with immobilized lipase in supercritical CO2 and reported its contribution in reducing the interaction between methanol and enzyme, reducing its toxicity [75] and immediately separating CO2 from the product.

New heterogeneous catalysts that can be easily recovered, regenerated and reused have been developed for biodiesel production. Such catalysts include alkaline earth metal oxides such as CaO and MgO, hydrotalcite, acid zirconia and alumina-based catalysts and immobilized lipase [20]. The use of a new nano catalyst consisting of CaO/CuFe2O4 during the transesterification process was successfully assayed for biodiesel production from chicken fat [69]. A sodium silicate catalyst that does not saponify with free fatty acids during transesterification was recently assayed to produce biodiesel that could be blended up to 30% with diesel, giving good performance. The brake specific fuel was 26% higher than diesel and the brake thermal efficiency was 4% lower while CO was reduced by 24.4% and hydrocarbons by 22.9%. However, no emission was increased by 11% at full load [25]. Shells of *Mytilus galloprovincialis*—waste from fish industry—containing CaO that can be used as a catalyst, were used for transesterification of jojoba oil. As CaO could be contaminated with CO2 and H2O, it was calcined immediately before use [126]. Calcined scallop shell was also reported as a very active catalyst for transesterification of rapeseed oil [127].

Recently, a cheap and safe catalyst consisting of metal hydrated salts was proposed for the pretreatment of animal fats with a high content of free fatty acids that could be esterified up to 99% with alcohol under mild conditions [128]. The methyl esters remained in the oily phase and could be used for transesterification directly with alkaline catalysts. On the other hand, a biorefining strategy for animal fat waste was proposed for the conversion of free fatty acids into triglycerides that could be blended with fossil diesel and be used in engine combustion systems [129]. Recently, adsorbents like magnesium aluminum hydroxycarbonate and 1,3,5-trimethyl-2,4,6-tris(3,5-ditert-butyl-4-hydroxybenzyl) benzene were proposed to enhance the oxidative stability of biodiesel and its blends and therefore retard their degradation. The acid value could be reduced up to 9%. In this way, the adsorbents can remove the precursors of the aging of biodiesel by stabilizing the generated free radicals and preventing them from starting new oxidation chains [130]. Precipitates of steryl glucosides are found in biodiesel produced from vegetable oils and may produce filter blockage. Their removal is achieved through adsorption with 3% silica at 112 ◦C for 72 min [131]. Finally, it is important to mention that Neste renewable diesel is produced through the hydrogen catalyzed conversion of triglycerides into the corresponding alkanes and propane. Nearly 3 million tons are produced in five plants and mixed with fossil diesel for its use in aviation, turbines, generators and ships [132].

### **6. Conclusions**

Biodiesel produced from agricultural waste has been rapidly expanded around the world due to its relevant advantages such as being biodegradable, renewable and sulphur-free. The cost of biodiesel majorly depends on the cost of the raw materials being used, with animal fat waste being cheaper than vegetable oil waste. Animal fats, usually found as waste from slaughterhouses, the meat processing industry, and cooking facilities, can be used as feedstock for biodiesel production. As reported in this manuscript, there are numerous processes already available for the production of biodiesel from animal fats and operating conditions during transesterification. There are also alternative solutions for pretreatment which mainly depend on the moisture and FFA content of such residues. Alkaline catalysis is still preferred at industrial plants producing biodiesel because it is faster than acid catalysis and cheaper than most alternative catalysts including acid catalysts and lipases. Although much research has been published on heterogeneous and acid and enzyme catalysts which avoid the challenges of water and FFA in animal fats, biodiesel producers have not yet adopted these technologies. However, there are recent developments that are promising for future industrial use. This is the case with heterogeneous catalysts that can be easily recovered, regenerated and reused, and immobilized lipases that improve efficiency and reduce costs by increasing enzyme stability and making the enzyme more resistant to denaturation by alcohol.

**Author Contributions:** Conceptualization, F.T.; resources, F.T.-R. and L.M.; writing—original draft preparation, F.T.-R.; writing—review and editing, F.T.-R., L.M. and F.T.; supervision, F.T. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by European Marie Curie project, grant number 614281 (HIGHVALFOOD) and European Regional Development Fund.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*
