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Modeling the supercritical carbon dioxide inactivation of Staphylococcus aureus, Escherichia coli and Bacillus subtilis in human body fluids clinical waste
Chemical Engineering Journal 296 (2016) 173–181
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Modeling the supercritical carbon dioxide inactivation of Staphylococcus aureus, Escherichia coli and Bacillus subtilis in human body fluids clinical waste Md. Sohrab Hossain a,⇑, N.A. Nik Norulaini b,⇑, Adel A. Banana c, A.R. Mohd Zulkhairi a, A.Y. Ahmad Naim a, A.K. Mohd Omar d a
Universiti Kuala Lumpur, Malaysian Institute of Chemical & Bioengineering Technology, 78000 Alor Gajah, Malacca, Malaysia School of Distance Education, Universiti Sains Malaysia, 11800 Penang, Malaysia c Environment Engineering Department, Subrata College, University of Zawia, Zawia, Libya d Department of Environmental Technology, School of Industrial Technology, Universiti Sains Malaysia, 11800 Penang, Malaysia b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
SC-CO2 is an efficient method for the
SC-CO2 inactivation of bacteria in human blood waste. (a) Human blood waste, (b) isolation of bacteria from human blood waste, (c) SC-CO2 sterilizer, (d) SEM image of SC-CO2 treated bacteria.
bacterial inactivation in human blood waste. SC-CO2 inactive bacteria by physicochemically degrade the cytoplasmic materials. SC-CO2 sterilization could use in a healthcare facility to improve hospital hygiene.
a r t i c l e
i n f o
Article history: Received 14 January 2016 Received in revised form 23 March 2016 Accepted 24 March 2016 Available online 30 March 2016 Keywords: Human blood waste Infectious waste Pathogenic bacteria Sterilization Supercritical carbon dioxide Waste management
a b s t r a c t Human body fluids clinical waste poses a challenge to healthcare facilities because of the presence of infectious pathogenic microorganisms, leading concern for an effective sterilization method to eliminate the infectious threat for safe handling and disposal. In the present study, the supercritical carbon dioxide (SC-CO2) was utilized to sterilize human blood waste with varying pressure and temperature for a treatment time of 5–90 min. Modified Gompertz equation was employed to elucidate Staphylococcus aureus (S. aureus), Escherichia coli (E. coli) and Bacillus subtilis (B. subtilis-vegetative cell) inactivation curve in SC-CO2 sterilized human blood waste. It was observed that the experimental data was well fitted with the predicted value obtained from modified Gompertz equation. The SC-CO2 sterilization efficiency was compared with the steam autoclave treatment based on bacterial regrowth potential and scanning electron microscope image analyzes. The absence of bacterial regrowth and physicochemical destruction of bacterial cells revealed that SC-CO2 is an efficient sterilization technology to treat human blood waste. Thus, SC-CO2 sterilization method could be utilized to sterilize human blood waste in a healthcare facility to improve hospital hygiene by eliminating infectious exposure of human body fluids clinical waste, and to conduct safe handling and management of human body fluids clinical waste. Ó 2016 Elsevier B.V. All rights reserved.
⇑ Corresponding authors. Tel.: +60 5512155; fax: +60 5512001 (Md. S. Hossain). Tel.: +60 4 6535206; fax: +60 4 6585435 (N.A. Nik Norulaini). E-mail addresses: [email protected] (M.S. Hossain), [email protected] (N.A. Nik Norulaini). http://dx.doi.org/10.1016/j.cej.2016.03.120 1385-8947/Ó 2016 Elsevier B.V. All rights reserved.
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1. Introduction There is increasing demand to determine an efficient sterilization technology in clinical waste management because of concern for hospital hygiene and environmental pollution [1,2]. Human body fluids clinical waste prescribed as a most infectious clinical waste due to contains nosocomial and opportunistic human pathogen, including bacteria, viruses, fungi and prions [3–5]. Human body fluids clinical waste is such a waste, which is in a liquid or semi-liquid states, such as blood, semen, vaginal secretions, synovial fluid, cerebral spinal fluid, pericardial fluid, pleural fluid, amniotic fluid, peritoneal fluid, and saliva from dental procedures [6,7]. Other human body fluids clinical waste are materials visibly contaminated with blood and in the situations of fluids when it is hard to differentiate between body fluids. Among the various types of body fluids clinical waste, human blood waste is gaining particular concern because of its enormous generation and the presence of beneficial nutrients for pathogen growth [4,8]. Typically, human blood is containing about 92% water, 8% plasma proteins and trace amounts of other materials. The technologies utilized at present to dispose human body fluids clinical waste are not environmentally friendly and unable to cope with clinical waste in a safe manner. Incineration is the most widely used technology in clinical waste management. However, incineration is viewed as unsuitable technology to dispose human blood waste due to the potential release of pollutants to the environment, high capital investment and occupational start-up cost requirements for the incineration facilities [6,8,9]. Moreover, incineration is not suitable to treat human blood waste due to moisture content over 30% [6,8]. Therefore, human blood waste is disposing in a sanitary swear after it has steam autoclaved or chemically disinfected [7,10]. Chemical disinfection of human blood waste is a process of the disinfecting pathogen in blood waste using chemicals, such as sodium hypochlorite, formaldehyde, chlorine dioxide and ozone gas [1,11,12]. Although the chemical disinfection can effectively control the microbial inactivation, there is a concern about the detrimental effects of chemical exposure to the aquatic environment [11,13]. The steam autoclave is typically operated at 121 °C for 15 min to decontaminate blood waste, based on the past application of steam sterilization of medical devices [12,14]. Studies reported that temperature and time of steam sterilization method might vary with waste type, composition and density [14–16]. Thus, the standard operating condition of steam autoclave (121 °C for 15 min) might not allow proper treatment of human blood waste [16]. Supercritical carbon dioxide (SC-CO2) has emerged as a nonthermal sterilization technology in various fields due to its low critical temperature. Pressurized CO2 beyond its critical point pressure (7.41 MPa) and temperature (31.1 °C), referred to as SC-CO2, is an effective sterilization method that offers notable benefit over existing technologies [17–20]. The sterilization efficiency of SCCO2 results from it physicochemical properties of fluids CO2, including its high dissolving power, high diffusivity and low viscosity [17,18,20]. The fluid CO2 in a supercritical state neither gas nor a liquid, which affect the pathogen in both physically and chemically. Numerous studies have conducted to inactive pathogens including viruses, fungi and bacteria and bacterial spores and found effective for that purpose [19–23]. However, few studies have been conducted to determine SC-CO2 mediated sterilization clinical waste management. In the previous studies, SC-CO2 was utilized to inactive pathogenic bacteria in clinical solid waste [18,19,21]. It was found that SC-CO2 effectively inactive the bacteria by destroying cell wall (physical effect of pressure) and denaturing the cytoplasmic materials (chemical effect of CO2) [15]. Further studies to evaluate the full extent of SC-CO2 sterilization
efficiency are vital to gain a better knowledge of this method so that it can be utilized in healthcare facilities to sterilize various types of clinical waste. Bacteria are unicellular microorganisms, can grow and survive indefinitely in a favorable environment and nutrient requirement (i.e., temperature, moisture, protein, fate nitrogen, phosphorus, oxygen) [24–26]. In a favorable environment, the bacteria can regrow and multiply extremely rapidly; the population can be double as quickly as every 9.8 min [26]. Thermal base treatment processes sterilize the clinical waste using heat. The heat treatment can affect the bacteria in clinical waste as inactive (destroyed the cytoplasmic materials and genomic DNA due to homogenous heat penetration), injured (not lethally damage the DNA due to heterogeneous heat penetration) and a fraction of bacteria escape from the heat penetration [18,27]. Subsequently, the injured bacteria may regrow in the nutrient rich clinical waste by repairing the injured DNA by photoreactivation processes and/or dark process [27]. The regrowth scenario of bacteria can be described as ‘‘viable but not cultivable”, as these injured and escape bacteria are not able colony forming on the surface agar media for a period until repaired DNA [28]. The main two factors, those are responsible for bacterial regrowth in sterilized clinical waste are (i) the growth of injured bacteria (ii) the reactivation of escape bacteria from heat penetration. Although studies have demonstrated the clinical waste sterilization using autoclave, microwave and chemical disinfectants, there is little information on the regrowth of bacteria in post-sterilized clinical waste. Therefore, it must be ensured the complete inactivation of the bacteria at the cellular level to avoid unexpected re-growth of bacteria prior to decide any sterilization technology for clinical waste [15]. In the present study, SC-CO2 was utilized to inactive Staphylococcus aureus, Escherichia coli and Bacillus subtilis in human blood waste. S. aureus is a gram-positive coccal bacterium. It is responsible for the most staphylococcal infections. S. aureus can cause severe and life-threatening infectious diseases including pneumonia, meningitis, osteomyelitis, endocarditis, bacteremia, and sepsis [29]. This bacteria (S. aureus) can survive on dry environmental surfaces for an hour to several months, depending on the stain. E. coli is gram negative bacterium, can cause severe food poisoning. These bacteria were chosen in the present study because S. aureus and E. coli are detected in clinical waste; wherein B. subtilis is high heat resistance gram-positive bacteria. The modified Gompertz equation was employed to elucidate the sigmoidal tendencies of microbial inactivation. The sterilization efficiency of SC-CO2 was compared with steam autoclave treatment using scanning electron microscope (SEM) image analysis and regrowth potential of bacteria in treated human blood waste. The findings of the present study will be useful to determine a suitable method for the human blood waste disposal.
2. Materials and methods 2.1. Sample collection and preparation Human blood used in the present study was collected using sterile blood collection tube from the pathological lab of General Hospital Pulau Pinang, Malaysia. The blood waste collected was autoclave at 121 °C for 15 min to eliminate possible contamination and ensure the safe handling. S. aureus, E. coli and B. subtilis used in the present study were taken from bacterial glycerol stock, which was isolated from clinical waste [4]. The isolated bacteria were reculture to gain fresh culture using blood agar media for S. aureus and B. subtilis; MacConkey agar media for E. coli. Subsequently, single isolated bacterial colony was inoculated in 20 mL autoclaved
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blood and incubated at 37 °C for 24 h. The septum of the cap of the tube was removed and replaced with two-layer filter to allow CO2 react with blood during treatment as well as preventing pathogenic bacteria from escaping.
phase, exponential inactivation phase and asymptotic phase. The modified Gompertz equation is expressed as follows [19,30]:
2.2. Treatment of human blood waste using SC-CO2
where log N/N0 is the logarithm ratio of viable cell count. A (log Nmax) is the lower asymptote value, kdm is the maximum inactivation rate (min1), k is the time for lag phase and t is the treatment time. The time length for the complete inactivation is defined by tt. At complete inactivation time, log N/N0 = A, with the tangent through the inflection point. Thus, the tt can be calculated as:
The experimental set-up for the SC-CO2 sterilizer is shown in Fig. 1. It consisted of a pump (American Lewa, Holistic, and Massachusetts, USA) with a maximum capacity of 68.9 MPa, an oven, a chiller (Yih Der, B/L-730), sterilization cell (volume 1.2 L) and liquefied carbon dioxide gas cylinder (65 °C). After replacing the sample in the SC-CO2 sterilization vessel, the vessel was tightly closed. The sensitivity of bacteria to the SC-CO2 was conducted with varying pressure (20–40 MPa) and temperature (30–60 °C) at treatment time of 5 min to 90 min. The SC-CO2 sterilization of human blood waste was performed following similar procedure as described elsewhere [19]. After treatment, the SC-CO2 treated sample was collected for the enumeration of the viable colony. Triplicate experiments were conducted and the results are expressed as means ± standard errors. 2.3. Enumeration of viable colony The number of viable colonies in the sample was determined before treatment (at the time 0 min) and after the treatment (at the time t min) using a pour-plate method. For the colony counting of bacteria, 1 mL of contaminated blood was taken for the eight fold serial dilution, from which 0.1 mL of removed for seeding on Petri dishes containing nutrient agar media using a sterile Drigalski spatula. The dishes with the culture medium were labeled and incubated at 37 °C for 24 h prior to counting. This procedure was carried out in duplicate to determine the average bacterial colony concentration in the blood waste. Results were expressed as the logarithm of surviving colony forming units per gram of waste (log CFU g1). The initial concentration of bacteria was found to be 7.14 ± 0.05 log CFU mL1, 6.81 ± 0.1 log CFU mL1 and 1 7.61 ± 0.1 log CFU mL for S. aureus, B. subtilis and E. coli, respectively. The log survival ratio of the number of viable colonies in per gram sample was calculated as the number of survival colonies after sterilization (N) to the number of viable colonies before treatment (N0). 2.4. Mathematical model for inactivation of bacteria using SC-CO2 The modified Gompertz equation was used to describe the sigmoidal curve for the bacterial inactivation, which includes lag
log
N kdm ¼ A: exp exp ðk tÞ þ 1 N0 A
tt ¼ k þ
A kdm
ð2Þ
The biological parameters (A, k, kdm) of the modified Gompertz model were determined using the experimental data. Whereas, the tt value was determined by following Eq. (2). The modified Gompertz equation (Eq. (1)) is a nonlinear equation; thereafter the bacteria cell inactivation data were fitted through non-linear regression with Levenberg–Marquardt algorithm in order to minimize the residual sum of the squares (RSS) of the difference between the predicted and observed value. Origin pro 6.1 (OriginLab Corporation, USA) was used to calculate the starting values by searching of the steepest ascent of the curve between the datum points (estimation of kdm), the line with the x-axis (estimation of k), and final datum point as the estimation for asymptote (A). The fitting goodness of the model was assessed using the regression coefficients (R2) and RSS values of the experimental and the predicted values at their 95% confidence level. 2.5. Scanning electron microscope image analysis of bacteria The bacterial contaminated blood waste was treated using steam autoclave (121 °C for 15 min) and SC-CO2 (20 MPa and 60 °C for 60 min). Treated and untreated bacteria contaminated human blood waste was centrifuged at 2000g for 15 min and the pellet of bacteria was collected. The collected pellet was suspended with McDowell-Trump fixative for 2 h. The cell was then rinsed 3 times using 0.1 M sodium phosphate buffer at pH 7.2 for 10 min. The suspended cells were post-fixed using 1% Osmium tetraoxide for 1 h. The cells were then rinsed 3 times using 0.1 M sodium phosphate buffer for 10 min each. The rinsed cells were dehydrated using a series of ethanol solution (50–100%) for 10 min each. Subsequently, the cells were rinsed 2 times with Hexamethyldisiazane 2 times for 10 min each and placed into a Desiccator to air dry at room temperature. The dried cells were then mounted into the SEM stub, coated with gold powder with double
Open/Close
Liquid CO2
Chiller 0~ 100C
Cooler DP: 10 MPa DT: 400C
CO2 Pump DP: 60 MPa
Sterilization Vessel
Band Heater 1kW
V01
ð1Þ
Preheater DP: 60 MPa DT: 800C
V02
V03
Fig. 1. Supercritical fluid carbon dioxide sterilization system. V, valve; DP, Design Pressure; DT, Design Temperature; V02 and V03, decompression valve.
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side carbon tape, and viewed using scanning electron microscope (VeriosTM XHR-450, Japan).
a
2
20 MPa (exp) 30 MPa (exp) 40 MPa (exp) 20 MPa (cal) 30 MPa (cal) 40 MPa (cal)
0
2.6. Re-growth of S. aureus, B. subtilis and E. coli in sterilized human blood waste Log (N/No )
The bacterial contaminated blood waste was sterilized using steam autoclave (121 °C for 15 min) and SC-CO2 (20 MPa and 60 °C for 60 min). After sterilization, the treated blood waste was stored at room temperature (25 ± 1 °C). The regrowth potential of bacteria in treated blood waste was determined at 24 h interval for 10 d. One milliliter of contaminate blood was taken and cultured on agar media. Blood and MacConkey agar media was used for the gram positive and gram negative bacteria.
-2
-4
-6
-8
-10 0
20
40
60
80
100
Pressure, MPa
3. Results and discussion 3.1. Sterilization human blood waste using SC-CO2
20 MPa (exp) 30 MPa (exp) 40 MPa (exp) 20 MPa (cal) 30 MPa (cal) 40 MPa (cal)
-2
0
Log (N/N )
0
-4
-6
-8 0
20
40
60
80
100
Pressure, MPa
c
2
20 MPa (exp) 30 MPa (exp) 40 MPa (exp) 20 MPa (cal) 30 MPa (cal) 40 MPa (cal)
0
-2
Log (N/N0 )
The influence of SC-CO2 pressure and temperature on the inactivation of E. coli, S. aureus and B. subtilis in human blood waste revealed that the bacterial inactivation curves were divided into the three distinct phases, such as lag phase, exponential phase, and stationary phases. The lag phase occurred at the initial stage of the bacterial inactivation due to SC-CO2 required some time to contact the bacteria. Bacterial inactivation reached its maximum at the end of the exponential phase, and therefore, the stationary phase occurred. To elucidate the actual microbial inactivation kinetics, the reduction in viable cell count of the studied bacteria was determined and fitted with the modified Gompertz equation. Fig. 2 shows the influence of SC-CO2 pressure on the inactivation of E. coli, S. aureus and B. subtilis in human blood waste. It was observed that the survivable ratios of E. coli, S. aureus, and B. subtilis decreased with increasing SC-CO2 pressure from 20 to 40 MPa at a constant temperature of 40 °C (Fig. 2). Moreover, the time required for complete inactivation of bacteria was considerably reduced with increasing pressure. At 20 MPa, it was required 60 min for the complete inactivation of E. coli and S. aureus; 75 min for the complete inactivation of B. subtilis. With the increase of pressure from 20 to 40 MPa, the complete inactivation time reduced to 30 min, 30 min and 45 min for the inactivation of E. coli, S. aureus, and B. subtilis, respectively. Table 1 shows the estimated values of A (stationary phase), k (lag phase), Kdm (inactivation rate), and tt (complete inactivation time) for the inactivation of E. coli, S. aureus and B. subtilis in human blood waste at various SC-CO2 pressure. The obtained regression coefficient (R2) values were greater than 0.93, suggesting a good fit between the experimental data and the calculated values from the modified Gompertz equation. The calculated tt values were close to the obtained experimental values for the complete inactivation of E. coli, S. aureus and B. subtilis in SC-CO2-treated human blood waste. Fig. 3 shows the inactivation curve of E. coli, S. aureus and B. subtilis in human blood waste at near critical (at 30 °C) and supercritical state (40–60 °C) of the fluid CO2. It was observed that the viable cell ratios decreased with increasing temperatures between 30 °C and 60 °C at a constant SC-CO2 pressure of 20 MPa. While survival ratio of the bacterial colony decreased with increasing temperature, the required complete inactivation time (time required to reached to 1 log CFU g-1) substantially reduced. The time necessary for the complete inactivation of E. coli and S. aureus were 75 min at 30 °C. Wherein, the complete inactivation time reduced to 30 min with increasing temperature from 30 °C to 60 °C. In the case of B. subtilis, the temperature 30 °C was unable to reduce the viable cell ratio at 1 log CFU g1. The required complete inactivation time was 90 min at temperature 40 °C, which
b
2
-4
-6
-8 0
20
40
60
80
100
Pressure, MPa
Fig. 2. Effect of pressure on the inactivation of bacteria in human blood waste using SC-CO2 at temperature 60 °C. (a) E. coli (b) S. aureus (c) B. subtilis; exp, experimental data; cal, calculated value by fitting the modified Gompertz equation to the experimental data.
reduced to 60 min at 60 °C. The estimated kinetic parameter values from the modified Gompertz equation for the inactivation of E. coli, S. aureus and B. subtilis in human blood waste are presented in Table 2. The R2 values were greater than 0.95, indicating an excellent fit of the experimental data to the calculated values obtained from the modified Gompertz equation. The modified Gompertz equation adequately described the inactivation kinetics of E. coli, S. aureus and B. subtilis in human blood waste subjected to SC-CO2 pressure and temperature (Figs. 2 and 3). The kdm values for the inactivation of E. coli, S. aureus and B. subtilis increased with increasing pressure and temperature. Both
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M.S. Hossain et al. / Chemical Engineering Journal 296 (2016) 173–181 Table 1 Estimation of the kinetics parameters of modified Gompertz equation for the inactivation of S. aureus, E. coli and B. subtilis at various SC-CO2 pressure. Pressure MPa
S. aureus
E. coli
A
kdm min1
k min
20 30 40
7.16 7.18 7.06
0.123 0.185 0.219
5..21 3.87 2.14
tt min
R
RSS
A
kdm min1
k min
63.42 42.69 34.38
0.96 0.98 0.93
0.28 1.53 1.66
7.68 7.72 7.69
0.152 0.241 0.405
4.36 3.26 1.27
a
2
o
30 C (exp) o 40 C (exp) o 50 C (exp) o 60 C (exp) o 30 C (cal) o 40 C (cal) o 50 C (cal) o 60 C (cal)
0
-2
Log (N/N0)
B. subtilis
2
-4
-6
-8
-10 0
20
40
60
80
100
Time, min
b
2
o
30 C (exp) o 40 C (exp) o 50 C (exp) o 60 C (exp) o 30 C (cal) o 40 C (cal) o 50 C (cal) o 60 C (cal)
Log (N/N0)
0
-2
tt min
R
2
RSS
A
kdm min1
k min
tt min
R2
RSS
54.88 35.29 20.25
0.95 0.97 0.98
1.05 0.32 1.19
6.81 6.79 6.84
0.094 0.149 0.164
6.2 5.36 3.27
78.64 50.93 44.98
0.95 0.98 0.97
0.65 1.22 1.55
which facilitated the CO2 to penetrate into the bacterial cell. The temperature simulates the diffusivity of CO2 and increases the fluidity of the bacterial cell [31,32]. Thus, the rise in the kdm value with increasing SC-CO2 pressure and temperature were due to the increase the solubility of CO2 and fluidity of the bacterial cell, which facilitated the penetration of CO2 into the bacteria cell easier and inactivated the bacteria at shorter treatment time [30]. State and Territorial Association in Alternative Treatment Technologies (STAATT) reported that the microbial inactivation slandered in clinical waste for an acceptable alternative technology of incineration should be 6 log reduction or greater [33]. As can see in Tables 1 and 2, SC-CO2 sterilization gained over 7 log reduction for S. aureus and E. coli; over 6 log reduction for B. subtilis at various combination of pressure, temperature and treatment time. Banana et al. [34] investigated the microwave inactivation of bacteria in human blood waste at 385, 450 and 700 W for 1–10 min. It was observed that Bacillus spp. inactivation was unable to reach 6 log reduction in microwave treated human blood waste. Thus, SC-CO2 sterilization technology can be considered as an acceptable alternative technology of human blood waste. 3.2. Analysis of temperature dependence by using the Arrhenius model The dependence of temperature at 20 MPa on the inactivation rate (kdm) of E. coli, S. aureus, and B. subtilis in response to SC-CO2 sterilized human blood was analyzed using the Arrhenius equation as below:
-4
-6
-8 0
20
40
60
80
100
Ed
kdm ¼ a:e RT
Time, min
ln kdm ¼ ln a þ
c
2
0
Log (N/N0)
Ed 1 T R
ð4Þ
o
30 C (exp) o 40 C (exp) o 50 C (exp) o 60 C (exp) o 30 C (cal) o 40 C (cal) o 50 C (cal) o 60 C (cal)
-2
-4
-6
-8 0
ð3Þ
20
40
60
80
100
Time, min
Fig. 3. Effect of temperature on the inactivation of bacteria in human blood waste using SC-CO2 at pressure 20 MPa. (a) E. coli (b) S. aureus (c) B. subtilis; exp, experimental data; cal, calculated value by fitting the modified Gompertz equation to the experimental data.
pressure and temperature influenced E. coli, S. aureus and B. subtilis inactivation efficiency in human blood waste by dominating cytoplasmic materials and mass transfer kinetics. The SC-CO2 pressure enhanced the solubilization with the moisture present in the human blood waste and acidified the bacterial cell membrane,
where T is the absolute temperature (K). Ed is the activation energy (kJ mol1), a is the pre-exponential factor (min1), and R is the molar gas constant (8.314 J mol1 K1). The activation energy (Ed) value reveals the temperature sensitivity of E. coli, S. aureus and B. subtilis to the SC-CO2 sterilization of human blood waste. Good fits of Ed values for the inactivation of E. coli, S. aureus and B. subtilis were gained by the Arrhenius equation, as shown in Fig. 4. The Ed values were 24.74 kJ mol1, 17.79 kJ mol1 and 12.78 kJ mol1 for the inactivation of E. coli, S. aureus and B. subtilis in SC-CO2 sterilized blood waste. The Ed values of E. coli and S. aureus determined in the present study were slightly higher than the Ed values of E. coli (14.95 kJ mol1) and S. aureus (11.61 kJ mol1) in clinical solid waste at SC-CO2 pressure of 10 MPa. Thus, the Ed values of bacterial inactivation depend on sample type tested [19]. The lower Ed values of E. coli, S. aureus and B. subtilis in SC-CO2-treated human blood waste implied that the inactivation of bacteria in SC-CO2 sterilization mostly depends on the pressure than the temperature during the sterilization process. Further, the Ed values for autoclave treated and thermally treated E. coli were found to be 96.17 kJ mol1 [18] and 532 kJ mol1 [35], respectively. The minimal Ed values for the inactivation of bacteria in blood waste using SC-CO2 with compare to thermal treatment method reveals that the SC-CO2 is a nonthermal sterilization technology to inactive bacteria in human
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Table 2 Estimation of the kinetics parameters of modified Gompertz equation for the inactivation of S. aureus, E. coli and B. subtilis at various SC-CO2 temperature. Temperature °C
S. aureus
E. coli
A
kdm min1
k min
tt min
R
RSS
A
kdm min1
k min
tt min
R
RSS
A
kdm min1
k min
tt min
R2
RSS
30 40 50 60
7.09 7.16 7.08 7.11
0.086 0.123 0.151 0.162
8.32 5.21 5.04 4.17
90.76 63.42 51.92 48.06
0.99 0.96 0.98 0.96
0.35 0.22 0.38 0.26
7.64 7.68 7.61 7.76
0.126 0.152 0.178 0.316
5.16 4.36 4.17 3.64
65.79 54.88 46.92 28.19
0.97 0.95 0.91 0.99
0.64 1.16 0.48 1.38
6.99 6.81 6.92 6.88
0.076 0.094 0.104 0.122
8.57 6.2 5.52 3.28
100.24 78.64 72.05 59.67
0.97 0.95 0.97 0.96
0.29 0.65 0.53 0.77
2.95E-03 -0.5
2
B. subtilis
3.05E-03
3.15E-03
2
3.25E-03
3.35E-03
E. coli S. aureus
-1.0
B. sublis
lnkdm (min-1)
y = -2975.8x + 7.6748 R² = 0.9201
Linear (E. coli)
-1.5
Linear (S. aureus) Linear (B. sublis) -2.0
-2.5
y = -1537.2x + 2.5127 R² = 0.9854 y = -2140.8x + 4.6754 R² = 0.9297
-3.0
1/T (K-1)
Fig. 4. Temperature dependence of bacteria in the inactivation of E. coli, S. aureus and B. subtilis in SC-CO2 treated human blood waste.
blood waste. Although, temperature is an important variable of SCCO2 sterilization, the influence temperature in SC-CO2 sterilization is not as high as in thermal treatment [30].
3.3. Analyze the morphological alteration of autoclaved and SC-CO2treated bacteria in human blood waste The morphological alternation of S. aureus, B. subtilis and E. coli in steam autoclaved and SC-CO2 treated human blood waste were determined using Scanning electron micrographs (SEM) analyzes, as shown in Fig. 5. Healthcare facilities are used to decontaminate the highly infectious clinical waste using steam autoclave at operating condition of 121 °C for 15 min before storage. In the present study, it was noted that the required time for the complete inactivation of S. aureus, B. subtilis and E. coli in SC-CO2 treated blood waste was 60 min at 20 MPa and 60 °C. Therefore, human blood waste was treated at temperature 121 °C for treatment 15 min for steam autoclave; pressure 20 MPa and temperature 60 °C for treatment 60 min for SC-CO2 to determine the inactivation mechanisms of S. aureus, B. subtilis and E. coli in treated human blood waste. It was observed that untreated and steam autoclave treated S. aureus, B. subtilis and E. coli cells were found to have smooth and uniform structural configurations. Conversely, SC-CO2 treated S. aureus, B. subtilis and E. coli cells showed broken cell walls, rupture, distortion, punctured holes, and loss of cellular membranes. The findings of the present study differ from for the study conducted by Dillow et al. [31]. Dillow et al. [31] observed that the minimal change of E. coli and S. aureus cells in biodegradable polymers subjected to SC-CO2 pressure of 20.5 MPa and temperature of 34 °C for
45 min. Kim et al. [30] found that the bacterial cells were almost unchanged, when analyzed SEM images of SC-CO2-treated and untreated E. coli O157:H7 at a pressure of 10 MPa, temperature 35 °C for 30 min. The significant morphological alteration in SCCO2-treated bacteria detected in this study could be attributed to the used of higher pressure, temperature, and time conditions [19]. The morphological changes in SC-CO2-treated bacterial were might due to the penetration of fluid CO2 and denature the cytoplasmic materials during SC-CO2 sterilization [30].
3.4. Bacterial regrowth in steam autoclaved and SC-CO2 treated clinical solid waste Table 3 shows the regrowth of S. aureus, B. subtilis and E. coli in steam autoclave and SC-CO2 treated human blood waste. No bacterial regrowth was detected in SC-CO2-treated human blood waste at post-sterilization day 0–10. Conversely, bacteria were found to regrow in steam autoclave treated human blood waste. The regrowth of B. subtilis in autoclaved blood waste started at poststerilization day 2, where S. aureus E. coli were found to regrow at and at day 3 and day 4, respectively. The absence of regrowth in SC-CO2 treated blood waste because of SC-CO2 inactivates bacteria by both physically (by pressure) and chemically (the fluids CO2 extracts the cytoplasmic materials). As a result, intact cytoplasmic materials of the bacteria cell are unlikely to remain in SC-CO2 treated blood waste to re-grow in the nutrient-rich human blood waste. Conversely, bacterial regrowth in steam autoclaved blood waste might be due to the incomplete destruction of bacteria during the sterilization. The sterilization efficiency of Steam autoclave
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b
a
500 nm
5000 nm
d
c
500 nm
500 0 nm
f
e
500 nm
5000 nm
g
h
1µ µm
1 µm
i
1 µm
Fig. 5. Scanning electron microscope image of untreated S. aureus, (a) autoclave treated S. aureus, (b) SC-CO2 treated S. aureus; untreated B. subtilis, (d) autoclave treated B. subtilis, (e) SC-CO2 treated B. subtilis; (f) untreated E. coli, (g) autoclave treated E. coli, (h) SC-CO2 treated E. coli (i).
Table 3 Re-growth of S. aureus, E. coli and B. subtilis in steam autoclave and SC-CO2 treated human blood waste. Treatment
Name of bacteria
Steam autoclave
SC-CO2
, re-growth negative;
p
Post sterilization time, day 0
1
2
S. aureus E. coli B. subtilis
p
S. aureus E. coli B. subtilis
, re-growth positive.
3 p p
4 p p p
5 p p p
6 p p p
7 p p p
8 p p p
9 p p p
10 p p p
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process depends on homogeneous heat penetration into the waste [10]. The saturated steam heat might denature the cytoplasmic materials (protein or enzymatic activity) and the bacteria inactivated. Some of the bacteria might injure or reach to the reversible stage due to the heterogeneous heat penetration into the waste. Subsequently, the injured bacteria cells re-grew in the presence of nutrients sufficient for cellular metabolism in human blood waste [25]. Similarly, Hossain et al. [14] observed that bacterial regrowth in steam autoclave treated clinical solid waste. Adel et al. [34] observed the regrowth of pathogenic bacteria in microwave-treated blood waste. However, regrowth of bacteria in sterilized sample have been observed by many researchers. For instance, Lee et al. [13] observed regrowth of coliforms bacteria in UV sterilized wastewater effluent. Shuval et al. [36] observed the regrowth of coliforms and fecal coliforms bacteria in chlorinated wastewater effluent. The finding of the present study reveals that SC-CO2 technology can be considered as an effective sterilization method for human blood waste, as SC-CO2 technology can inactive bacteria in cellular level. Thus, it is bearing considerable interest to set up this technology in a healthcare facility to treat human blood waste before discharge in the sewage system. The adoption of SC-CO2 technology in human body fluids clinical waste treatment would benefit the healthcare facility in several ways including reduce infectious exposure, improve hospital hygiene and conduct safe handling and management of human body fluids clinical waste.
4. Conclusion The modified Gompertz equation well described the SC-CO2 inactivation of S. aureus, B. subtilis and E. coli in human blood waste. Both pressure and temperature had a potential effect on S. aureus, B. subtilis and E. coli inactivation in human blood waste. The Arrhenius model illustrated that the SC-CO2 inactivation of S. aureus, B. subtilis and E. coli in human blood waste was less dependent on SC-CO2 sterilization temperature than the SC-CO2 pressure. Analyzes of the SEM image of untreated, steam autoclave treated and SC-CO2 treated S. aureus, B. subtilis and E. coli cells reveals that SC-CO2 inactive the bacteria physicochemically (breaking the cell wall by SC-CO2 pressure) and chemically (denature the cytoplasmic materials by the fluids CO2). Analyzes of the bacterial regrowth potential in a steam autoclave and SC-CO2 treated human blood waste reveals that steam autoclave is an inappropriate method to treat human blood waste. Conversely, the absence of regrowth in SC-CO2 treated human blood waste indicated that SC-CO2 is an effective way to inactivate bacteria in human blood waste. The finding of the present study strongly suggests sterilizing the human blood waste at its generation source (healthcare facilities) using SC-CO2 before discharge into the sewage system. The adoption of SC-CO2 technology in human body fluids clinical waste treatment in a healthcare facility would reduce infectious exposure, improve hospital hygiene and conduct safe handling and management of human body fluids clinical waste.
Acknowledgements We would like to give due thanks and appreciation to the staff of the Department of Microbiology, Penang General Hospital, Penang, Malaysia for their technical assistance, unceasing cooperation and support during the entire study period. Also, the authors would like to express their appreciations to Universiti Sains Malaysia, Penang, Malaysia for the financial assistance to this research via a research grants (203/PTEKIND/6711438) and (1002/ PJJAUH/910324).
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Chemical Engineering Journal journal homepage: www.elsevier.com/locate/cej
Modeling the supercritical carbon dioxide inactivation of Staphylococcus aureus, Escherichia coli and Bacillus subtilis in human body fluids clinical waste Md. Sohrab Hossain a,⇑, N.A. Nik Norulaini b,⇑, Adel A. Banana c, A.R. Mohd Zulkhairi a, A.Y. Ahmad Naim a, A.K. Mohd Omar d a
Universiti Kuala Lumpur, Malaysian Institute of Chemical & Bioengineering Technology, 78000 Alor Gajah, Malacca, Malaysia School of Distance Education, Universiti Sains Malaysia, 11800 Penang, Malaysia c Environment Engineering Department, Subrata College, University of Zawia, Zawia, Libya d Department of Environmental Technology, School of Industrial Technology, Universiti Sains Malaysia, 11800 Penang, Malaysia b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
SC-CO2 is an efficient method for the
SC-CO2 inactivation of bacteria in human blood waste. (a) Human blood waste, (b) isolation of bacteria from human blood waste, (c) SC-CO2 sterilizer, (d) SEM image of SC-CO2 treated bacteria.
bacterial inactivation in human blood waste. SC-CO2 inactive bacteria by physicochemically degrade the cytoplasmic materials. SC-CO2 sterilization could use in a healthcare facility to improve hospital hygiene.
a r t i c l e
i n f o
Article history: Received 14 January 2016 Received in revised form 23 March 2016 Accepted 24 March 2016 Available online 30 March 2016 Keywords: Human blood waste Infectious waste Pathogenic bacteria Sterilization Supercritical carbon dioxide Waste management
a b s t r a c t Human body fluids clinical waste poses a challenge to healthcare facilities because of the presence of infectious pathogenic microorganisms, leading concern for an effective sterilization method to eliminate the infectious threat for safe handling and disposal. In the present study, the supercritical carbon dioxide (SC-CO2) was utilized to sterilize human blood waste with varying pressure and temperature for a treatment time of 5–90 min. Modified Gompertz equation was employed to elucidate Staphylococcus aureus (S. aureus), Escherichia coli (E. coli) and Bacillus subtilis (B. subtilis-vegetative cell) inactivation curve in SC-CO2 sterilized human blood waste. It was observed that the experimental data was well fitted with the predicted value obtained from modified Gompertz equation. The SC-CO2 sterilization efficiency was compared with the steam autoclave treatment based on bacterial regrowth potential and scanning electron microscope image analyzes. The absence of bacterial regrowth and physicochemical destruction of bacterial cells revealed that SC-CO2 is an efficient sterilization technology to treat human blood waste. Thus, SC-CO2 sterilization method could be utilized to sterilize human blood waste in a healthcare facility to improve hospital hygiene by eliminating infectious exposure of human body fluids clinical waste, and to conduct safe handling and management of human body fluids clinical waste. Ó 2016 Elsevier B.V. All rights reserved.
⇑ Corresponding authors. Tel.: +60 5512155; fax: +60 5512001 (Md. S. Hossain). Tel.: +60 4 6535206; fax: +60 4 6585435 (N.A. Nik Norulaini). E-mail addresses: [email protected] (M.S. Hossain), [email protected] (N.A. Nik Norulaini). http://dx.doi.org/10.1016/j.cej.2016.03.120 1385-8947/Ó 2016 Elsevier B.V. All rights reserved.
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1. Introduction There is increasing demand to determine an efficient sterilization technology in clinical waste management because of concern for hospital hygiene and environmental pollution [1,2]. Human body fluids clinical waste prescribed as a most infectious clinical waste due to contains nosocomial and opportunistic human pathogen, including bacteria, viruses, fungi and prions [3–5]. Human body fluids clinical waste is such a waste, which is in a liquid or semi-liquid states, such as blood, semen, vaginal secretions, synovial fluid, cerebral spinal fluid, pericardial fluid, pleural fluid, amniotic fluid, peritoneal fluid, and saliva from dental procedures [6,7]. Other human body fluids clinical waste are materials visibly contaminated with blood and in the situations of fluids when it is hard to differentiate between body fluids. Among the various types of body fluids clinical waste, human blood waste is gaining particular concern because of its enormous generation and the presence of beneficial nutrients for pathogen growth [4,8]. Typically, human blood is containing about 92% water, 8% plasma proteins and trace amounts of other materials. The technologies utilized at present to dispose human body fluids clinical waste are not environmentally friendly and unable to cope with clinical waste in a safe manner. Incineration is the most widely used technology in clinical waste management. However, incineration is viewed as unsuitable technology to dispose human blood waste due to the potential release of pollutants to the environment, high capital investment and occupational start-up cost requirements for the incineration facilities [6,8,9]. Moreover, incineration is not suitable to treat human blood waste due to moisture content over 30% [6,8]. Therefore, human blood waste is disposing in a sanitary swear after it has steam autoclaved or chemically disinfected [7,10]. Chemical disinfection of human blood waste is a process of the disinfecting pathogen in blood waste using chemicals, such as sodium hypochlorite, formaldehyde, chlorine dioxide and ozone gas [1,11,12]. Although the chemical disinfection can effectively control the microbial inactivation, there is a concern about the detrimental effects of chemical exposure to the aquatic environment [11,13]. The steam autoclave is typically operated at 121 °C for 15 min to decontaminate blood waste, based on the past application of steam sterilization of medical devices [12,14]. Studies reported that temperature and time of steam sterilization method might vary with waste type, composition and density [14–16]. Thus, the standard operating condition of steam autoclave (121 °C for 15 min) might not allow proper treatment of human blood waste [16]. Supercritical carbon dioxide (SC-CO2) has emerged as a nonthermal sterilization technology in various fields due to its low critical temperature. Pressurized CO2 beyond its critical point pressure (7.41 MPa) and temperature (31.1 °C), referred to as SC-CO2, is an effective sterilization method that offers notable benefit over existing technologies [17–20]. The sterilization efficiency of SCCO2 results from it physicochemical properties of fluids CO2, including its high dissolving power, high diffusivity and low viscosity [17,18,20]. The fluid CO2 in a supercritical state neither gas nor a liquid, which affect the pathogen in both physically and chemically. Numerous studies have conducted to inactive pathogens including viruses, fungi and bacteria and bacterial spores and found effective for that purpose [19–23]. However, few studies have been conducted to determine SC-CO2 mediated sterilization clinical waste management. In the previous studies, SC-CO2 was utilized to inactive pathogenic bacteria in clinical solid waste [18,19,21]. It was found that SC-CO2 effectively inactive the bacteria by destroying cell wall (physical effect of pressure) and denaturing the cytoplasmic materials (chemical effect of CO2) [15]. Further studies to evaluate the full extent of SC-CO2 sterilization
efficiency are vital to gain a better knowledge of this method so that it can be utilized in healthcare facilities to sterilize various types of clinical waste. Bacteria are unicellular microorganisms, can grow and survive indefinitely in a favorable environment and nutrient requirement (i.e., temperature, moisture, protein, fate nitrogen, phosphorus, oxygen) [24–26]. In a favorable environment, the bacteria can regrow and multiply extremely rapidly; the population can be double as quickly as every 9.8 min [26]. Thermal base treatment processes sterilize the clinical waste using heat. The heat treatment can affect the bacteria in clinical waste as inactive (destroyed the cytoplasmic materials and genomic DNA due to homogenous heat penetration), injured (not lethally damage the DNA due to heterogeneous heat penetration) and a fraction of bacteria escape from the heat penetration [18,27]. Subsequently, the injured bacteria may regrow in the nutrient rich clinical waste by repairing the injured DNA by photoreactivation processes and/or dark process [27]. The regrowth scenario of bacteria can be described as ‘‘viable but not cultivable”, as these injured and escape bacteria are not able colony forming on the surface agar media for a period until repaired DNA [28]. The main two factors, those are responsible for bacterial regrowth in sterilized clinical waste are (i) the growth of injured bacteria (ii) the reactivation of escape bacteria from heat penetration. Although studies have demonstrated the clinical waste sterilization using autoclave, microwave and chemical disinfectants, there is little information on the regrowth of bacteria in post-sterilized clinical waste. Therefore, it must be ensured the complete inactivation of the bacteria at the cellular level to avoid unexpected re-growth of bacteria prior to decide any sterilization technology for clinical waste [15]. In the present study, SC-CO2 was utilized to inactive Staphylococcus aureus, Escherichia coli and Bacillus subtilis in human blood waste. S. aureus is a gram-positive coccal bacterium. It is responsible for the most staphylococcal infections. S. aureus can cause severe and life-threatening infectious diseases including pneumonia, meningitis, osteomyelitis, endocarditis, bacteremia, and sepsis [29]. This bacteria (S. aureus) can survive on dry environmental surfaces for an hour to several months, depending on the stain. E. coli is gram negative bacterium, can cause severe food poisoning. These bacteria were chosen in the present study because S. aureus and E. coli are detected in clinical waste; wherein B. subtilis is high heat resistance gram-positive bacteria. The modified Gompertz equation was employed to elucidate the sigmoidal tendencies of microbial inactivation. The sterilization efficiency of SC-CO2 was compared with steam autoclave treatment using scanning electron microscope (SEM) image analysis and regrowth potential of bacteria in treated human blood waste. The findings of the present study will be useful to determine a suitable method for the human blood waste disposal.
2. Materials and methods 2.1. Sample collection and preparation Human blood used in the present study was collected using sterile blood collection tube from the pathological lab of General Hospital Pulau Pinang, Malaysia. The blood waste collected was autoclave at 121 °C for 15 min to eliminate possible contamination and ensure the safe handling. S. aureus, E. coli and B. subtilis used in the present study were taken from bacterial glycerol stock, which was isolated from clinical waste [4]. The isolated bacteria were reculture to gain fresh culture using blood agar media for S. aureus and B. subtilis; MacConkey agar media for E. coli. Subsequently, single isolated bacterial colony was inoculated in 20 mL autoclaved
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blood and incubated at 37 °C for 24 h. The septum of the cap of the tube was removed and replaced with two-layer filter to allow CO2 react with blood during treatment as well as preventing pathogenic bacteria from escaping.
phase, exponential inactivation phase and asymptotic phase. The modified Gompertz equation is expressed as follows [19,30]:
2.2. Treatment of human blood waste using SC-CO2
where log N/N0 is the logarithm ratio of viable cell count. A (log Nmax) is the lower asymptote value, kdm is the maximum inactivation rate (min1), k is the time for lag phase and t is the treatment time. The time length for the complete inactivation is defined by tt. At complete inactivation time, log N/N0 = A, with the tangent through the inflection point. Thus, the tt can be calculated as:
The experimental set-up for the SC-CO2 sterilizer is shown in Fig. 1. It consisted of a pump (American Lewa, Holistic, and Massachusetts, USA) with a maximum capacity of 68.9 MPa, an oven, a chiller (Yih Der, B/L-730), sterilization cell (volume 1.2 L) and liquefied carbon dioxide gas cylinder (65 °C). After replacing the sample in the SC-CO2 sterilization vessel, the vessel was tightly closed. The sensitivity of bacteria to the SC-CO2 was conducted with varying pressure (20–40 MPa) and temperature (30–60 °C) at treatment time of 5 min to 90 min. The SC-CO2 sterilization of human blood waste was performed following similar procedure as described elsewhere [19]. After treatment, the SC-CO2 treated sample was collected for the enumeration of the viable colony. Triplicate experiments were conducted and the results are expressed as means ± standard errors. 2.3. Enumeration of viable colony The number of viable colonies in the sample was determined before treatment (at the time 0 min) and after the treatment (at the time t min) using a pour-plate method. For the colony counting of bacteria, 1 mL of contaminated blood was taken for the eight fold serial dilution, from which 0.1 mL of removed for seeding on Petri dishes containing nutrient agar media using a sterile Drigalski spatula. The dishes with the culture medium were labeled and incubated at 37 °C for 24 h prior to counting. This procedure was carried out in duplicate to determine the average bacterial colony concentration in the blood waste. Results were expressed as the logarithm of surviving colony forming units per gram of waste (log CFU g1). The initial concentration of bacteria was found to be 7.14 ± 0.05 log CFU mL1, 6.81 ± 0.1 log CFU mL1 and 1 7.61 ± 0.1 log CFU mL for S. aureus, B. subtilis and E. coli, respectively. The log survival ratio of the number of viable colonies in per gram sample was calculated as the number of survival colonies after sterilization (N) to the number of viable colonies before treatment (N0). 2.4. Mathematical model for inactivation of bacteria using SC-CO2 The modified Gompertz equation was used to describe the sigmoidal curve for the bacterial inactivation, which includes lag
log
N kdm ¼ A: exp exp ðk tÞ þ 1 N0 A
tt ¼ k þ
A kdm
ð2Þ
The biological parameters (A, k, kdm) of the modified Gompertz model were determined using the experimental data. Whereas, the tt value was determined by following Eq. (2). The modified Gompertz equation (Eq. (1)) is a nonlinear equation; thereafter the bacteria cell inactivation data were fitted through non-linear regression with Levenberg–Marquardt algorithm in order to minimize the residual sum of the squares (RSS) of the difference between the predicted and observed value. Origin pro 6.1 (OriginLab Corporation, USA) was used to calculate the starting values by searching of the steepest ascent of the curve between the datum points (estimation of kdm), the line with the x-axis (estimation of k), and final datum point as the estimation for asymptote (A). The fitting goodness of the model was assessed using the regression coefficients (R2) and RSS values of the experimental and the predicted values at their 95% confidence level. 2.5. Scanning electron microscope image analysis of bacteria The bacterial contaminated blood waste was treated using steam autoclave (121 °C for 15 min) and SC-CO2 (20 MPa and 60 °C for 60 min). Treated and untreated bacteria contaminated human blood waste was centrifuged at 2000g for 15 min and the pellet of bacteria was collected. The collected pellet was suspended with McDowell-Trump fixative for 2 h. The cell was then rinsed 3 times using 0.1 M sodium phosphate buffer at pH 7.2 for 10 min. The suspended cells were post-fixed using 1% Osmium tetraoxide for 1 h. The cells were then rinsed 3 times using 0.1 M sodium phosphate buffer for 10 min each. The rinsed cells were dehydrated using a series of ethanol solution (50–100%) for 10 min each. Subsequently, the cells were rinsed 2 times with Hexamethyldisiazane 2 times for 10 min each and placed into a Desiccator to air dry at room temperature. The dried cells were then mounted into the SEM stub, coated with gold powder with double
Open/Close
Liquid CO2
Chiller 0~ 100C
Cooler DP: 10 MPa DT: 400C
CO2 Pump DP: 60 MPa
Sterilization Vessel
Band Heater 1kW
V01
ð1Þ
Preheater DP: 60 MPa DT: 800C
V02
V03
Fig. 1. Supercritical fluid carbon dioxide sterilization system. V, valve; DP, Design Pressure; DT, Design Temperature; V02 and V03, decompression valve.
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side carbon tape, and viewed using scanning electron microscope (VeriosTM XHR-450, Japan).
a
2
20 MPa (exp) 30 MPa (exp) 40 MPa (exp) 20 MPa (cal) 30 MPa (cal) 40 MPa (cal)
0
2.6. Re-growth of S. aureus, B. subtilis and E. coli in sterilized human blood waste Log (N/No )
The bacterial contaminated blood waste was sterilized using steam autoclave (121 °C for 15 min) and SC-CO2 (20 MPa and 60 °C for 60 min). After sterilization, the treated blood waste was stored at room temperature (25 ± 1 °C). The regrowth potential of bacteria in treated blood waste was determined at 24 h interval for 10 d. One milliliter of contaminate blood was taken and cultured on agar media. Blood and MacConkey agar media was used for the gram positive and gram negative bacteria.
-2
-4
-6
-8
-10 0
20
40
60
80
100
Pressure, MPa
3. Results and discussion 3.1. Sterilization human blood waste using SC-CO2
20 MPa (exp) 30 MPa (exp) 40 MPa (exp) 20 MPa (cal) 30 MPa (cal) 40 MPa (cal)
-2
0
Log (N/N )
0
-4
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-8 0
20
40
60
80
100
Pressure, MPa
c
2
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0
-2
Log (N/N0 )
The influence of SC-CO2 pressure and temperature on the inactivation of E. coli, S. aureus and B. subtilis in human blood waste revealed that the bacterial inactivation curves were divided into the three distinct phases, such as lag phase, exponential phase, and stationary phases. The lag phase occurred at the initial stage of the bacterial inactivation due to SC-CO2 required some time to contact the bacteria. Bacterial inactivation reached its maximum at the end of the exponential phase, and therefore, the stationary phase occurred. To elucidate the actual microbial inactivation kinetics, the reduction in viable cell count of the studied bacteria was determined and fitted with the modified Gompertz equation. Fig. 2 shows the influence of SC-CO2 pressure on the inactivation of E. coli, S. aureus and B. subtilis in human blood waste. It was observed that the survivable ratios of E. coli, S. aureus, and B. subtilis decreased with increasing SC-CO2 pressure from 20 to 40 MPa at a constant temperature of 40 °C (Fig. 2). Moreover, the time required for complete inactivation of bacteria was considerably reduced with increasing pressure. At 20 MPa, it was required 60 min for the complete inactivation of E. coli and S. aureus; 75 min for the complete inactivation of B. subtilis. With the increase of pressure from 20 to 40 MPa, the complete inactivation time reduced to 30 min, 30 min and 45 min for the inactivation of E. coli, S. aureus, and B. subtilis, respectively. Table 1 shows the estimated values of A (stationary phase), k (lag phase), Kdm (inactivation rate), and tt (complete inactivation time) for the inactivation of E. coli, S. aureus and B. subtilis in human blood waste at various SC-CO2 pressure. The obtained regression coefficient (R2) values were greater than 0.93, suggesting a good fit between the experimental data and the calculated values from the modified Gompertz equation. The calculated tt values were close to the obtained experimental values for the complete inactivation of E. coli, S. aureus and B. subtilis in SC-CO2-treated human blood waste. Fig. 3 shows the inactivation curve of E. coli, S. aureus and B. subtilis in human blood waste at near critical (at 30 °C) and supercritical state (40–60 °C) of the fluid CO2. It was observed that the viable cell ratios decreased with increasing temperatures between 30 °C and 60 °C at a constant SC-CO2 pressure of 20 MPa. While survival ratio of the bacterial colony decreased with increasing temperature, the required complete inactivation time (time required to reached to 1 log CFU g-1) substantially reduced. The time necessary for the complete inactivation of E. coli and S. aureus were 75 min at 30 °C. Wherein, the complete inactivation time reduced to 30 min with increasing temperature from 30 °C to 60 °C. In the case of B. subtilis, the temperature 30 °C was unable to reduce the viable cell ratio at 1 log CFU g1. The required complete inactivation time was 90 min at temperature 40 °C, which
b
2
-4
-6
-8 0
20
40
60
80
100
Pressure, MPa
Fig. 2. Effect of pressure on the inactivation of bacteria in human blood waste using SC-CO2 at temperature 60 °C. (a) E. coli (b) S. aureus (c) B. subtilis; exp, experimental data; cal, calculated value by fitting the modified Gompertz equation to the experimental data.
reduced to 60 min at 60 °C. The estimated kinetic parameter values from the modified Gompertz equation for the inactivation of E. coli, S. aureus and B. subtilis in human blood waste are presented in Table 2. The R2 values were greater than 0.95, indicating an excellent fit of the experimental data to the calculated values obtained from the modified Gompertz equation. The modified Gompertz equation adequately described the inactivation kinetics of E. coli, S. aureus and B. subtilis in human blood waste subjected to SC-CO2 pressure and temperature (Figs. 2 and 3). The kdm values for the inactivation of E. coli, S. aureus and B. subtilis increased with increasing pressure and temperature. Both
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M.S. Hossain et al. / Chemical Engineering Journal 296 (2016) 173–181 Table 1 Estimation of the kinetics parameters of modified Gompertz equation for the inactivation of S. aureus, E. coli and B. subtilis at various SC-CO2 pressure. Pressure MPa
S. aureus
E. coli
A
kdm min1
k min
20 30 40
7.16 7.18 7.06
0.123 0.185 0.219
5..21 3.87 2.14
tt min
R
RSS
A
kdm min1
k min
63.42 42.69 34.38
0.96 0.98 0.93
0.28 1.53 1.66
7.68 7.72 7.69
0.152 0.241 0.405
4.36 3.26 1.27
a
2
o
30 C (exp) o 40 C (exp) o 50 C (exp) o 60 C (exp) o 30 C (cal) o 40 C (cal) o 50 C (cal) o 60 C (cal)
0
-2
Log (N/N0)
B. subtilis
2
-4
-6
-8
-10 0
20
40
60
80
100
Time, min
b
2
o
30 C (exp) o 40 C (exp) o 50 C (exp) o 60 C (exp) o 30 C (cal) o 40 C (cal) o 50 C (cal) o 60 C (cal)
Log (N/N0)
0
-2
tt min
R
2
RSS
A
kdm min1
k min
tt min
R2
RSS
54.88 35.29 20.25
0.95 0.97 0.98
1.05 0.32 1.19
6.81 6.79 6.84
0.094 0.149 0.164
6.2 5.36 3.27
78.64 50.93 44.98
0.95 0.98 0.97
0.65 1.22 1.55
which facilitated the CO2 to penetrate into the bacterial cell. The temperature simulates the diffusivity of CO2 and increases the fluidity of the bacterial cell [31,32]. Thus, the rise in the kdm value with increasing SC-CO2 pressure and temperature were due to the increase the solubility of CO2 and fluidity of the bacterial cell, which facilitated the penetration of CO2 into the bacteria cell easier and inactivated the bacteria at shorter treatment time [30]. State and Territorial Association in Alternative Treatment Technologies (STAATT) reported that the microbial inactivation slandered in clinical waste for an acceptable alternative technology of incineration should be 6 log reduction or greater [33]. As can see in Tables 1 and 2, SC-CO2 sterilization gained over 7 log reduction for S. aureus and E. coli; over 6 log reduction for B. subtilis at various combination of pressure, temperature and treatment time. Banana et al. [34] investigated the microwave inactivation of bacteria in human blood waste at 385, 450 and 700 W for 1–10 min. It was observed that Bacillus spp. inactivation was unable to reach 6 log reduction in microwave treated human blood waste. Thus, SC-CO2 sterilization technology can be considered as an acceptable alternative technology of human blood waste. 3.2. Analysis of temperature dependence by using the Arrhenius model The dependence of temperature at 20 MPa on the inactivation rate (kdm) of E. coli, S. aureus, and B. subtilis in response to SC-CO2 sterilized human blood was analyzed using the Arrhenius equation as below:
-4
-6
-8 0
20
40
60
80
100
Ed
kdm ¼ a:e RT
Time, min
ln kdm ¼ ln a þ
c
2
0
Log (N/N0)
Ed 1 T R
ð4Þ
o
30 C (exp) o 40 C (exp) o 50 C (exp) o 60 C (exp) o 30 C (cal) o 40 C (cal) o 50 C (cal) o 60 C (cal)
-2
-4
-6
-8 0
ð3Þ
20
40
60
80
100
Time, min
Fig. 3. Effect of temperature on the inactivation of bacteria in human blood waste using SC-CO2 at pressure 20 MPa. (a) E. coli (b) S. aureus (c) B. subtilis; exp, experimental data; cal, calculated value by fitting the modified Gompertz equation to the experimental data.
pressure and temperature influenced E. coli, S. aureus and B. subtilis inactivation efficiency in human blood waste by dominating cytoplasmic materials and mass transfer kinetics. The SC-CO2 pressure enhanced the solubilization with the moisture present in the human blood waste and acidified the bacterial cell membrane,
where T is the absolute temperature (K). Ed is the activation energy (kJ mol1), a is the pre-exponential factor (min1), and R is the molar gas constant (8.314 J mol1 K1). The activation energy (Ed) value reveals the temperature sensitivity of E. coli, S. aureus and B. subtilis to the SC-CO2 sterilization of human blood waste. Good fits of Ed values for the inactivation of E. coli, S. aureus and B. subtilis were gained by the Arrhenius equation, as shown in Fig. 4. The Ed values were 24.74 kJ mol1, 17.79 kJ mol1 and 12.78 kJ mol1 for the inactivation of E. coli, S. aureus and B. subtilis in SC-CO2 sterilized blood waste. The Ed values of E. coli and S. aureus determined in the present study were slightly higher than the Ed values of E. coli (14.95 kJ mol1) and S. aureus (11.61 kJ mol1) in clinical solid waste at SC-CO2 pressure of 10 MPa. Thus, the Ed values of bacterial inactivation depend on sample type tested [19]. The lower Ed values of E. coli, S. aureus and B. subtilis in SC-CO2-treated human blood waste implied that the inactivation of bacteria in SC-CO2 sterilization mostly depends on the pressure than the temperature during the sterilization process. Further, the Ed values for autoclave treated and thermally treated E. coli were found to be 96.17 kJ mol1 [18] and 532 kJ mol1 [35], respectively. The minimal Ed values for the inactivation of bacteria in blood waste using SC-CO2 with compare to thermal treatment method reveals that the SC-CO2 is a nonthermal sterilization technology to inactive bacteria in human
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Table 2 Estimation of the kinetics parameters of modified Gompertz equation for the inactivation of S. aureus, E. coli and B. subtilis at various SC-CO2 temperature. Temperature °C
S. aureus
E. coli
A
kdm min1
k min
tt min
R
RSS
A
kdm min1
k min
tt min
R
RSS
A
kdm min1
k min
tt min
R2
RSS
30 40 50 60
7.09 7.16 7.08 7.11
0.086 0.123 0.151 0.162
8.32 5.21 5.04 4.17
90.76 63.42 51.92 48.06
0.99 0.96 0.98 0.96
0.35 0.22 0.38 0.26
7.64 7.68 7.61 7.76
0.126 0.152 0.178 0.316
5.16 4.36 4.17 3.64
65.79 54.88 46.92 28.19
0.97 0.95 0.91 0.99
0.64 1.16 0.48 1.38
6.99 6.81 6.92 6.88
0.076 0.094 0.104 0.122
8.57 6.2 5.52 3.28
100.24 78.64 72.05 59.67
0.97 0.95 0.97 0.96
0.29 0.65 0.53 0.77
2.95E-03 -0.5
2
B. subtilis
3.05E-03
3.15E-03
2
3.25E-03
3.35E-03
E. coli S. aureus
-1.0
B. sublis
lnkdm (min-1)
y = -2975.8x + 7.6748 R² = 0.9201
Linear (E. coli)
-1.5
Linear (S. aureus) Linear (B. sublis) -2.0
-2.5
y = -1537.2x + 2.5127 R² = 0.9854 y = -2140.8x + 4.6754 R² = 0.9297
-3.0
1/T (K-1)
Fig. 4. Temperature dependence of bacteria in the inactivation of E. coli, S. aureus and B. subtilis in SC-CO2 treated human blood waste.
blood waste. Although, temperature is an important variable of SCCO2 sterilization, the influence temperature in SC-CO2 sterilization is not as high as in thermal treatment [30].
3.3. Analyze the morphological alteration of autoclaved and SC-CO2treated bacteria in human blood waste The morphological alternation of S. aureus, B. subtilis and E. coli in steam autoclaved and SC-CO2 treated human blood waste were determined using Scanning electron micrographs (SEM) analyzes, as shown in Fig. 5. Healthcare facilities are used to decontaminate the highly infectious clinical waste using steam autoclave at operating condition of 121 °C for 15 min before storage. In the present study, it was noted that the required time for the complete inactivation of S. aureus, B. subtilis and E. coli in SC-CO2 treated blood waste was 60 min at 20 MPa and 60 °C. Therefore, human blood waste was treated at temperature 121 °C for treatment 15 min for steam autoclave; pressure 20 MPa and temperature 60 °C for treatment 60 min for SC-CO2 to determine the inactivation mechanisms of S. aureus, B. subtilis and E. coli in treated human blood waste. It was observed that untreated and steam autoclave treated S. aureus, B. subtilis and E. coli cells were found to have smooth and uniform structural configurations. Conversely, SC-CO2 treated S. aureus, B. subtilis and E. coli cells showed broken cell walls, rupture, distortion, punctured holes, and loss of cellular membranes. The findings of the present study differ from for the study conducted by Dillow et al. [31]. Dillow et al. [31] observed that the minimal change of E. coli and S. aureus cells in biodegradable polymers subjected to SC-CO2 pressure of 20.5 MPa and temperature of 34 °C for
45 min. Kim et al. [30] found that the bacterial cells were almost unchanged, when analyzed SEM images of SC-CO2-treated and untreated E. coli O157:H7 at a pressure of 10 MPa, temperature 35 °C for 30 min. The significant morphological alteration in SCCO2-treated bacteria detected in this study could be attributed to the used of higher pressure, temperature, and time conditions [19]. The morphological changes in SC-CO2-treated bacterial were might due to the penetration of fluid CO2 and denature the cytoplasmic materials during SC-CO2 sterilization [30].
3.4. Bacterial regrowth in steam autoclaved and SC-CO2 treated clinical solid waste Table 3 shows the regrowth of S. aureus, B. subtilis and E. coli in steam autoclave and SC-CO2 treated human blood waste. No bacterial regrowth was detected in SC-CO2-treated human blood waste at post-sterilization day 0–10. Conversely, bacteria were found to regrow in steam autoclave treated human blood waste. The regrowth of B. subtilis in autoclaved blood waste started at poststerilization day 2, where S. aureus E. coli were found to regrow at and at day 3 and day 4, respectively. The absence of regrowth in SC-CO2 treated blood waste because of SC-CO2 inactivates bacteria by both physically (by pressure) and chemically (the fluids CO2 extracts the cytoplasmic materials). As a result, intact cytoplasmic materials of the bacteria cell are unlikely to remain in SC-CO2 treated blood waste to re-grow in the nutrient-rich human blood waste. Conversely, bacterial regrowth in steam autoclaved blood waste might be due to the incomplete destruction of bacteria during the sterilization. The sterilization efficiency of Steam autoclave
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b
a
500 nm
5000 nm
d
c
500 nm
500 0 nm
f
e
500 nm
5000 nm
g
h
1µ µm
1 µm
i
1 µm
Fig. 5. Scanning electron microscope image of untreated S. aureus, (a) autoclave treated S. aureus, (b) SC-CO2 treated S. aureus; untreated B. subtilis, (d) autoclave treated B. subtilis, (e) SC-CO2 treated B. subtilis; (f) untreated E. coli, (g) autoclave treated E. coli, (h) SC-CO2 treated E. coli (i).
Table 3 Re-growth of S. aureus, E. coli and B. subtilis in steam autoclave and SC-CO2 treated human blood waste. Treatment
Name of bacteria
Steam autoclave
SC-CO2
, re-growth negative;
p
Post sterilization time, day 0
1
2
S. aureus E. coli B. subtilis
p
S. aureus E. coli B. subtilis
, re-growth positive.
3 p p
4 p p p
5 p p p
6 p p p
7 p p p
8 p p p
9 p p p
10 p p p
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process depends on homogeneous heat penetration into the waste [10]. The saturated steam heat might denature the cytoplasmic materials (protein or enzymatic activity) and the bacteria inactivated. Some of the bacteria might injure or reach to the reversible stage due to the heterogeneous heat penetration into the waste. Subsequently, the injured bacteria cells re-grew in the presence of nutrients sufficient for cellular metabolism in human blood waste [25]. Similarly, Hossain et al. [14] observed that bacterial regrowth in steam autoclave treated clinical solid waste. Adel et al. [34] observed the regrowth of pathogenic bacteria in microwave-treated blood waste. However, regrowth of bacteria in sterilized sample have been observed by many researchers. For instance, Lee et al. [13] observed regrowth of coliforms bacteria in UV sterilized wastewater effluent. Shuval et al. [36] observed the regrowth of coliforms and fecal coliforms bacteria in chlorinated wastewater effluent. The finding of the present study reveals that SC-CO2 technology can be considered as an effective sterilization method for human blood waste, as SC-CO2 technology can inactive bacteria in cellular level. Thus, it is bearing considerable interest to set up this technology in a healthcare facility to treat human blood waste before discharge in the sewage system. The adoption of SC-CO2 technology in human body fluids clinical waste treatment would benefit the healthcare facility in several ways including reduce infectious exposure, improve hospital hygiene and conduct safe handling and management of human body fluids clinical waste.
4. Conclusion The modified Gompertz equation well described the SC-CO2 inactivation of S. aureus, B. subtilis and E. coli in human blood waste. Both pressure and temperature had a potential effect on S. aureus, B. subtilis and E. coli inactivation in human blood waste. The Arrhenius model illustrated that the SC-CO2 inactivation of S. aureus, B. subtilis and E. coli in human blood waste was less dependent on SC-CO2 sterilization temperature than the SC-CO2 pressure. Analyzes of the SEM image of untreated, steam autoclave treated and SC-CO2 treated S. aureus, B. subtilis and E. coli cells reveals that SC-CO2 inactive the bacteria physicochemically (breaking the cell wall by SC-CO2 pressure) and chemically (denature the cytoplasmic materials by the fluids CO2). Analyzes of the bacterial regrowth potential in a steam autoclave and SC-CO2 treated human blood waste reveals that steam autoclave is an inappropriate method to treat human blood waste. Conversely, the absence of regrowth in SC-CO2 treated human blood waste indicated that SC-CO2 is an effective way to inactivate bacteria in human blood waste. The finding of the present study strongly suggests sterilizing the human blood waste at its generation source (healthcare facilities) using SC-CO2 before discharge into the sewage system. The adoption of SC-CO2 technology in human body fluids clinical waste treatment in a healthcare facility would reduce infectious exposure, improve hospital hygiene and conduct safe handling and management of human body fluids clinical waste.
Acknowledgements We would like to give due thanks and appreciation to the staff of the Department of Microbiology, Penang General Hospital, Penang, Malaysia for their technical assistance, unceasing cooperation and support during the entire study period. Also, the authors would like to express their appreciations to Universiti Sains Malaysia, Penang, Malaysia for the financial assistance to this research via a research grants (203/PTEKIND/6711438) and (1002/ PJJAUH/910324).
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