Bioaugmentation process of secondary effluents for reduction of pathogens, heavy metals and antibiotics

Proper handling of sewage effluents has gained heightened recognition due to the presence of many pollutants, including pathogenic bacteria, viruses and parasites, as well as heavy meals and antibiotics (Sano et al. 2003; Ji et al. 2012; Al-Gheethi et al. 2015). In fact, for many years, different techniques, such as adsorption, ion exchange, ozonation, UV irradiation and filtration, have been used to remove those contaminants from sewage effluents (Dutta et al. 1999; Koivunen et al. 2003; Dodd et al. 2006; Batt et al. 2007; Putra et al. 2009). Nevertheless, despite the numerous claims pertaining to their effectiveness, it was revealed that there are inherent setbacks in the application of these technologies, including insufficiency, as well as in producing toxic by-product and carcinogenic compounds, as in the case of the oxidation processes of ozonation and chlorination (Xu et al. 2002; Putra et al. 2009; Wang & Chen 2009; Vital et al. 2010; Wang et al. 2012).

In terms of the disinfection process, the oxidation process of ozonation has led to the increase of assimilable organic carbon, and thus, increases the pathogen growth potential, as recorded for Escherichia coli O157 and of Pseudomonas aeruginosa (Vital et al. 2010). In addition, the UV irradiation of secondary effluents might be insufficient due to the presence of high suspended solids that protect the bacterial cells from UV damage. Therefore, regrowth and resistance among these bacteria have been observed, such as the regrowth and the repair potential, as well as the resistance among E. coli, faecal coliform (FC), Bacillus subtilis and Clostridium sp. in disinfected waters (Alonso et al. 2004; Ting et al. 2011; Wang et al. 2012).

Also, in regard to heavy metals, both adsorption and ion exchange techniques have become ineffective in achieving the removal level acquired to meet the standard limits if the heavy metals’ concentrations are between 1 and 100 mg L⁻¹ (Putra et al. 2009; Wang & Chen 2009). These techniques are designed to remove one pollutant, such as heavy metals alone; therefore, the secondary effluents have to be subjected to further treatment process to reduce other pollutants, such as pathogenic bacteria. On the other hand, the antibiotics could not be removed completely by sewage treatment processes, and the removal efficiency ranged between −34 and 72% (Gao et al. 2012). β-lactam antibiotics enter sewage streams through faeces and urine because the main removal of β-lactam antibiotics from the body is by secretion into the urine (Pauwels & Verstraete 2006). In fact, the discovery of antibiotics in sewage effluents has been reported by numerous authors in the literature: trimethoprim and ciprofloxacin in Sweden (Olofsson 2004); erythromycin, tetracycline, cephalexin and norfloxacin in Hong Kong (Gulkowska et al. 2008); fleroxacin, ofloxacin, sulfapyridine and sulfamethoxazole in China (Gao et al. 2012); as well as amoxicillin, ampicillin and ciprofloxacin in Tanzania (Kihampa 2014).

As mentioned earlier, it appears that in order to remove more than one pollutant, such as pathogenic bacteria, heavy metals and antibiotics, a series of treatment stages is required. Nevertheless, the treatment process has to be a simple system with only a few requirements of chemical additives, besides possessing the ability to remove a wide range of the challenging contaminants within one stage (Lim et al. 2002).

In addition, the increased loads of antibiotics and heavy metals in sewage might induce the bacterial cells to acquire resistance/tolerances, either by mutation or by the transfer of resistant genes among the bacteria. This results in the bacterial strain to have the potential to resist antibiotics and heavy metals simultaneously (Silva et al. 2006). For instance, Bacillus spp. have developed strategies for survival in unfavourable environments. Other than that, many decomposing bacteria are present during sewage treatment in the stage concerning the biological process (secondary process). In this work, B. subtilis 2012WTNC, which was isolated from secondary effluents, displayed the potential to biodegrade cephalexin by β-lactamase. The ability of β-lactamase (EC 3.5.2.6) produced from bacteria to hydrolyse the β-lactam ring of β-lactam antibiotics, thus, had been found to further inactivate the compounds (Liao et al. 2010). Therefore, the bioaugmentation process, including adding selected indigenous bacteria strains into sewage effluents, might be efficient in degrading antibiotics, as well as in removing heavy metals via biodegradation and biosorption processes simultaneously. Moreover, bioaugmentation of wastewater by adding selected strains’ cultures can improve the catabolism of specific compounds and it is indeed a promising technique to solve practical problems in wastewater treatment plants, and to enhance removal efficiency (Herrero & Stuckey 2015). This process, nonetheless, would be more efficient if conducted at a temperature above that required for pathogenic bacterial growth, which does mean that this process would contribute in reducing pathogens.

For instance, a previous work that employed treatment of sewage at 45 °C exhibited high efficiency in reducing pathogenic bacteria (Al-Gheethi et al. 2013). B. subtilis had been found to be effective in removing antibiotics and heavy metals from sewage effluents at 37 °C (Al-Gheethi & Norli 2014; Adel et al. 2015). In this study, a strain of B. subtilis, which was grown during the thermal treatment of sewage, was applied as reference in a previous work (Al-Gheethi et al. 2013). This strain was found to be thermotolerant and exhibited resistance against heavy metals and antibiotics. Therefore, the bioaugmentation with the thermotolerant strain of B. subtilis was used in this study to remove heavy metals and β-lactam antibiotics from different types of secondary effluents in one stage, and this emphasizes the novelty of the current work. Faecal indicator (FC and Enterococci) and pathogenic bacteria (Staphylococcus aureus and Salmonella spp.) were reduced as a result of the thermal treatment conducted at 45 °C.

Moreover, Ni²⁺ ions and cephalexin were chosen as the model because previous work had found that there was a correlation between bacterial resistance against nickel and cephalexin (Al-Gheethi & Norli 2014). Furthermore, the cephalexin was resistant against degradation during the sewage treatment process, while nickel removal had been limited during the treatment process in comparison to other heavy metals (Leung et al. 2001). Chua & Hua (1996) found that the nickel concentration of 25 mg L⁻¹ caused serious upsets in the biological processes of the sewage treatment. This concentration could not be removed via chemical and physical techniques. As well, Ni²⁺ at low concentrations plays the role of cofactor for some bacterial enzymes, and a previous study had indicated that the production of β-lactamase increased in the presence of nickel ions (Al-Gheethi & Norli 2014).

Hence, the theoretical hypothesis for this treatment is given as follows: the treatment process conducted at 45 °C enhances the ability of B. subtilis 2012WTNC as a thermotolerant strain to grow, as well as reducing faecal indicators and pathogenic bacteria. The ability of B. subtilis 2012WTNC to tolerate cephalexin and to produce β-lactamase leads to the biodegradation of cephalexin and the production of bacterial biomass yield. Thus, the resulting biomass would remove nickel ions as a result of the affinity of metal ions towards bacterial surfaces (Abdel-Monem et al. 2010; Al-Gheethi et al. 2014).

The secondary effluents were collected from the Jelutong Sewage Treatment Plant (JSTP) located at Penang, Malaysia. The sewage that flowed to this plant comprised residential, commercial and institutional wastes. The plant was designed in 2008 and received 270,000 m³/day from 1.2 million population equivalent in 2014 (Saad & Zahera 2013, personal communication, Jelutong Sewage Treatment Plant, Penang, Malaysia). The JSTP was classified as a typical sewage treatment plant; the treatment of sewage in the plant was based on sequenced batch reactors (Saad & Zahera 2013, personal communication, Jelutong Sewage Treatment Plant, Penang, Malaysia). The waste treatment facility was designed to generate effluent quality to meet the EAQ 1974, regulation 2009 (sewage) Standard A requirements, which should be less than 20 mg L⁻¹ of BOD (biochemical oxygen demand), 50 mg L⁻¹ of TSS (total suspended solids) and 10 mg L⁻¹ of total ammonia nitrogen, plus nitrate nitrogen with a limit of 5 mg L⁻¹ of ammonia nitrogen. As well, the plant used UV irradiation for the disinfection process. However, the microbiological quality of secondary effluents was not determined before final disposal into the receiving sea water.

Glass bottles (1 L) used for the collection of samples were sterilized in an autoclave (high-pressure steam sterilization, Model ES-315, Tomy-Japan) at 15 psi and 121 °C for 15 min. The collection of samples (1 L) was carried out from the point of disposal of secondary effluents. For the heavy metals’ determination, 2.4 mL of a 15% solution of ethylene di-amine-tetra acetic acid tetra-sodium salt (R & M Marketing, Essex, UK), was added as a chelating agent to the samples. The samples were transported to the laboratory in a cooler box and the microbiological analysis was carried out within 2 h of collection.

Four secondary effluent samples were prepared in the laboratory, including secondary effluents (E1), secondary effluents containing 10 mg L⁻¹ (w/v) of Ni²⁺ ions (E2), secondary effluents containing 1 g L⁻¹ (w/v) of cephalexin (E3), and secondary effluents containing 10 mg L⁻¹ (w/v) of Ni²⁺ and 1 g L⁻¹ (w/v) of cephalexin (E4). These samples were prepared to represent different types of secondary effluents. The cephalexin was added into the secondary effluents with high concentrations to simulate pharmaceutical and hospital effluents due to the difficulty of obtaining these samples in the study area. The concentrations of cephalexin were added based on previous studies reported for antibiotics (Qiting & Xiheng 1988; Li et al. 2008). Furthermore, the nickel ions concentrations used in this study were less and could be removed by chemical technologies, such as adsorption and ion exchange processes, as mentioned above.

Additionally, FC, Enterococci, S. aureus and Salmonella spp., as well as enzymatic biodegradation and sorption processes in the secondary effluents treated at 45 °C, were evaluated. Sewage treated effluents without B. subtilis 2012WTNC were used as control. Moreover, in order to avoid false results, the concentration of Ni²⁺ ions metals in the original sewage treated effluents was determined and used as a blank. Another secondary effluent sample containing cephalexin without bacterial inoculum was also used as a blank.

One litre of each effluent sample (in triplicate) was placed in a glass bottle (1 L), inoculated with 10 mL (v/v) of B. subtilis 2012 WTNC (∼1.8 × 10⁶ CFU mL⁻¹). The effluents were incubated in an incubator (JE10 TECH, Model SI.300R-Korea) at 45 °C. The sample containing bottles were covered with aluminium foil to prevent photo-degradation of antibiotics in E3 and E4, while E1 and E2 were covered to provide equal treatment conditions for all samples. The samples were taken at days 0, 1, 2, 4, 8, 16, 32 and 64, whereby a fixed volume of 100 mL of each sample was taken for bacteriological, chemical and enzymatic analyses.

FC, Enterococci, S. aureus and Salmonella spp. were enumerated by using membrane filtration techniques (Sartorius, Germany) with a sterilized membrane filter (Millipore cellulose or nylon membrane filters; 45 mm diameter, 0.45 mm pore size) on the appropriate selective media. In brief, the sample bottle was shaken vigorously at least 25 times to distribute the bacteria uniformly. One mL of each effluent was filtered, and the membrane filter was removed from the filter base with forceps (sterilized by alcohol flaming) and placed onto the Endo agar for FC, bile esculin iron agar (BEIA) for Enterococci, xylose lysine deoxycholate agar (XLD) for Salmonella spp. and mannitol salt agar (MSA) for S. aureus. The plates were closed and sealed using a Para-film, then transferred into a plastic bag containing wet cotton to prevent dryness, and incubated for 24–48 h at 37 °C for E. faecalis and Salmonella spp., and S. aureus at 44.5 °C for FC.

The colonies of FC grew with a greenish metallic sheen or pink colour on the Endo-agar and EMB agar. In order to confirm the FC colonies, APHA (1999) was employed. In brief, a loopful of a single colony grown on the Endo agar and the EMB agar incubated at 44.5 °C was transferred and inoculated to an EC medium broth tube containing Durham tubes. After 24 h, the tubes were examined for growth and gas bubbles in the EC Medium (Merck, Germany) broth tube to confirm the presence of FC.

The colonies of E. faecalis grown on BEIA had brown centres, while the colonies of S. aureus on MSA grew with a gold colour, and the Salmonella spp. was red in colour with black centres. Salmonella spp., S. aureus and E. faecalis were identified based on their morphological and cultural characteristics, as well as biochemical tests by using API 20 Strep (Ref 07226 B) and API 20NE (Ref 07224 B) (BioMerieux, SA-France). Furthermore, a catalase test was performed by adding a drop of 3% H₂O₂ (BioMerieux, SA-France) to the bacterial slant tubes and oxygen bubbles indicated that the test was positive. Other than that, oxidase production was tested by using the wet filter paper methods with oxidase test reagents (BioMerieux, SA-France), where violet colour indicated that the test was positive.

Next, the number of bacteria was calculated and reported in terms of CFU of the bacteria per 100 mL of the sewage effluent sample. In order to ensure that the reduction of faecal indicators and pathogenic bacteria had been less than the detection limits, 10 mL of the treated effluent was filtrated through the membrane filtration and then, the filter was transferred and placed on the surface of a brain–heart infusion medium as an enriched medium and incubated at 37 °C for 48 h; the absence of growth indicated the total elimination of bacteria (Al-Gheethi et al. 2013). If there was no growth of bacteria, the number of colonies were considered as 10 CFU/100 mL.

The biodegradation of cephalexin by B. subtilis 2012WTNC was determined based on the determination of β-lactamase in the secondary effluents. β-lactamase (EC 3.5.2.6) was identified by determining the hydrolysis of cephalexin substrate according to Çelik & Çalik (2004). In brief, after the incubation period at intervals of 0, 1, 2, 4, 8, 16, 32 and 64 days, a fixed volume (10 mL) of the secondary effluent samples (E3 and E4) were harvested via centrifugation at 13,500 (rpm) for 10 min and the supernatant was tested for β-lactamase. The benzyl penicillin was replaced by cephalexin as substrate according to Sawai et al. (1978). Furthermore, the optimal wavelength of the cephalexin absorbance among different wavelengths was determined, and was 340 nm with a spectrophotometer (Model DR 2800, HACH, Germany (Appendix B, available with the online version of this paper)).The calibration curve of the different cephalexin concentrations in 0.1 M phosphate buffer had been determined at 30 °C and pH = 7.0 (λ = 340 nm) (Appendix C, available with the online version of this paper). In order to determine the β-lactamase concentrations in the secondary effluents, fresh substrate solutions were prepared daily by dissolving 0.4 mg mL⁻¹ (w/v) of cephalexin in 0.1 M phosphate buffer (pH 7) and maintained at 30 °C. A fixed volume (0.1 mL) of centrifuged culture supernatant was added to 3 mL (v/v) of substrate solution. Immediately after that, the changes in absorbance over 1 min were recorded.

The secondary effluent sample containing cephalexin without bacterial inoculum was used as a blank. One unit of β-lactamase concentration was defined as the amount of enzyme that could hydrolyse 1 μmol of cephalexin at 30 °C and pH 7.0 in 1 min. The product of the hydrolysis reaction, cephalosporanic acid, also displayed absorbance at 340 nm, and therefore, the difference of the slopes was y₁–y₂ = 0.2135 A_λ.

The concentrations of enzyme, U mL⁻¹, is given by 1U = A_λ × 0.2135 × Dilution factor.

The data were subjected to a one-way analysis of variance (ANOVA) with three determinations. The results were compared by employing the one-way ANOVA post hoc multiple comparison (LSD). The ANOVA tests were performed to determine the significance of the differences among the results. The differences were considered significant at p < 0.05.

In the present work, the bioaugmentation process for four types of secondary effluents using indigenous B. subtilis 2012WTNC were carried out at 45 °C. The efficiency of this process to reduce faecal indicators (FC and Enterococci) and pathogenic bacteria (Salmonella spp. and S. aureus), enzymatic biodegradation of cephalexin, as well as removal of Ni²⁺ ions, was determined. B. subtilis 2012WTNC was chosen based on specific criteria, which are described earlier and presented in Tables 1–5. B. subtilis 2012WTNC exhibited high growth at 45 °C than thermotolerant bacteria. Therefore, in addition to the effectiveness of the treatment process at 45 °C to reduce indicators and pathogenic bacteria, this process had successfully removed both heavy metals and antibiotics.

Table 3

Characteristics of Bacillus subtilis 2012WTNC (CP003492.1) obtained from secondary effluents. Tolerance of high concentrations of nickel ions and the association with the resistance to antibiotics

.				Sensitivity test .
Concentration of Ni2+ (mM) .				Control .					Tolerance at 2 mM of Ni2+ .					Tolerance at 4 mM of Ni2+ .					Tolerance at 6 mM of Ni2+ .
1 mM	2 mM	4 mM	6 mM
+ ++ +	+ ++ +	+ ++ +	+ ++	R	R	R	S	R	ND	ND	ND	R	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND

.				Sensitivity test .
Concentration of Ni2+ (mM) .				Control .					Tolerance at 2 mM of Ni2+ .					Tolerance at 4 mM of Ni2+ .					Tolerance at 6 mM of Ni2+ .
1 mM	2 mM	4 mM	6 mM
+ ++ +	+ ++ +	+ ++ +	+ ++	R	R	R	S	R	ND	ND	ND	R	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND

++ +, good; ++ + +, excellent. S = susceptible, diameter of the inhibition zone >1.2 cm; M = moderate, diameter of the inhibition zone between 1.0 and 1.2 cm; R = resistance, diameter of the inhibition zone was between 0.7 and 1.0 cm. ND, not detected if the bacteria has exhibited resistance for antibiotics.

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Comprehension of the growth requirements of pathogenic bacteria in aquatic ecosystems is essential for a holistic approach to microbial risk assessment, as well as for improving effluents’ treatment design and operation. In fact, several factors determine the growth of pathogenic bacteria in the aquatic environment. Vital et al. (2010) identified the environmental conditions and competition with the indigenous microbial flora as the main consideration in reducing pathogens in water systems.

Regarding an environment supporting bacterial growth, the strength of the treatment process in disinfecting secondary effluents depends on the inactivation of pathogenic bacteria and the constitution of the secondary effluents. It has been well-documented that pathogenic bacteria grow in a range between 10 and 40 °C and with the best growth at a moderate temperature or close to the human temperature (37 °C) (Frey & Price 2003). In this work, the treatment process was conducted at 45 °C. The retention time and the temperature are the most influential factors for microorganism removal (Alcalde et al. 2003). Lucero-Ramirez (2000) reported that pathogenic bacteria in sewage reduced to less than detectable levels in properly operated heat-drying systems.

Nonetheless, interestingly, the bacterial species investigated in this study exhibited different responses to the treatment process. Gram-positive bacteria (Enterococci and S. aureus) survived more than Gram-negative bacteria (FC and Salmonella spp.). Similar findings were also reported in previous work where E. faecalis exhibited more resistance than E. coli during the treatment process at different temperatures (55 and 65 °C). These differences are explained based on the cell wall structure. Gram-positive bacteria displayed a cell wall with greater strength than Gram-negative bacteria. Celico et al. (2004) also indicated that Enterococci are a more reliable indicator than FC for the detection of microbial pollution because Enterococci are more resistant in the environment than FC. In fact, Enterococci have been frequently considered as a reference microorganism for thermal treatments to be applied in pasteurized foods (Smith et al. 1990; Ghazala et al. 1991). However, WHO (1989) and US EPA (2004) did not regulate standard limits of Enterococci in secondary effluents. In addition, in comparison between FC and Salmonella spp. (both are Gram-negative bacteria), the results revealed that Salmonella spp. survived more than FC. It demonstrated that Salmonella spp. was resistant and projected the potential to survive under hostile environmental conditions (Espigares et al. 2006; Alvarez-Ordonez et al. 2011). Therefore, US EPA (1995) and US EPA (2003) suggested it as an indicator of choice for monitoring the effectiveness of sewage pathogen reduction. In contrast, Enterococci displayed more resistance than S. aureus (both are Gram-positive bacteria) during the treatment process. Enterococci showed the ability to grow at both 10 °C and 45 °C, at high pH (9.6) and in a medium containing 6.5% of NaCl (Cooper & Olivieri 1998; Carvalho & Teixeira 2002). Enterococcus is an environmental organism and it has been suggested as useful for indicating the presence of viruses, particularly in sludge, sea water and biosolids (Bitton 2005). S. aureus has been detected previously in sewage and hospital wastewater (Rusin et al. 2003; Thompson et al. 2013) and has been proposed as an indicator of hospital hygiene for microbiological standards (Dancer 2004). For instance, Jin et al. (2013) used S. aureus as an indicator to be evaluated in a hydrothermal treatment process for achievement of hygienic safety for food waste. US EPA (1988) classified this bacterium in the minor list, which concerns opportunistic pathogens that only cause disease in debilitated or immunologically compromised individuals, but the evaluation of the indicator value of S. aureus requires further studies.

It is worth noting that the reductions in faecal indicators and pathogenic bacteria were in response to the treatment process at 45 °C and not as a result of competition with B. subtilis 2012WTNC, where the reductions of faecal indicators and pathogenic bacteria in the control samples (without B. subtilis 2012WTNC) were quite similar and did not differ significantly in comparison to the bioaugmented ones. In previous work, the concentrations of faecal indicators and pathogenic bacteria were reduced to less than the detection limits after 1 week of the treatment process at 45 °C, without bioaugmentation with B. subtilis 2012WTNC (Al-Gheethi et al. 2013). Moreover, in this study, B. subtilis 2012WTNC had grown to be thermotolerant bacteria, while faecal indicators and pathogenic bacteria were reduced to less than the detection limits, as determined by using the culture-based method on the enrichment medium.

With regard to that, for the composition of the secondary effluents, it was observed that the reductions of FC and Enterococci in E3 were significantly more than those in E1, E2 and E4, as determined by the paired t-test (p < 0.05). The growth of B. subtilis 2012WTNC in E3 was also less than in E1, E2 and E4. This might be due to the presence of cephalexin, which inhibited the bacterial cell wall synthesis, thus enhancing the reduction levels. B. subtilis 2012WTNC was less sensitive to cephalexin as it was acclimatized to tolerate high concentrations of cephalexin during the screening process. E4 also contained cephalexin, but the presence of nickel ions might limit the effect of cephalexin on the bacterial cell growth.

As previously stated, it was noted that the presence of FC, Enterococci, Salmonella spp. and S. aureus of the secondary samples after 64 days of the bioaugmentation process were less than the detection limits, in comparison to the concentrations at zero day, which indicated that the quality of the secondary effluents had improved.

Microorganisms, in many ways, can take up heavy metals through biosorption on the bacterial surface or via bioaccumulation, where the metal crosses through the membrane to enter the bacterial cytoplasm (Brierley 1990). In the present work, it was found that the removal of Ni²⁺ ions by B. subtilis 2012WTNC took place within the period of 1 to 4 days via bioaccumulation, where the bacterial cells exhibited the maximum growth and accumulated nickel ions (Figure 6). The removal process after 8 days occurred through the biosorption process, where the biomass yield was at the maximum level (Figure 8). The removal of nickel ion in the E4 was less than in E2, 96.63 versus 66.33% (Figure 9). This may occur as the nickel ions were adsorbed to the functional group of cephalexin, insoluble in mixed effluents and formed complexes, and thus, inhibited the removal process by bacterial biomass, as in the case of Al²⁺ and Fe²⁺ oxides that formed ciprofloxacin–surface complexes (Gu & Karthikeyan 2008).

According to EQA 1974, Regulation 2009 (Sewage) Standards A and B, the pH value of secondary effluents should be in the range of 5–9. Therefore, the best treatment process is the one that does not change the pH of the treated effluents to less than 5 or more than 9. In this study, the pH was in the range of 5–9 at the end of the treatment process.

B. subtilis 2012WTNC displayed the potential to remove both β-lactam antibiotics and heavy metals from secondary effluents that had been heavily contaminated with heavy metals, as well as to confront any contamination in secondary effluents obtained from hospital sewage or industrial wastewaters. B. subtilis 2012WTNC was chosen in this study based on its specific criteria determined in the section ‘Bacterial strain’. These criteria were applied in this work and were suitable to choose the bacterial isolates to remove heavy metals from secondary effluents.

It can be concluded that the bioaugmentation process investigated in this study projected the potential to reduce faecal indicators and pathogenic bacteria, heavy metals, as well as β-lactam antibiotics in one stage. Two major treatment processes were combined into one stage, temperature control and the addition of B. subtilis. As a result, FC was reduced to meet the standard limits set by the US EPA, and nickel ions were decreased to meet EQA1974, Regulation 2009 (Sewage) Standard B. Other than that, the pH levels were still in the range recommended by these regulations. This process would be able to improve the quality of secondary effluents. The sewage treated effluents generated from this process were suitable for safe disposal or to be reused in agriculture. However, it is important to indicate that the process had been proven to be efficient for inactivation of pathogenic bacteria in those specified conditions. Moreover, more studies with other types of pathogens, such as viruses, should be carried out.

The authors gratefully acknowledge the Third World Organization for Academic Science (TWAS), Ministry of Higher Education of Malaysia for the research project financial support under fundamental research grant scheme (FRGS) No. 1453 and FRGS (203/PTEKIND/6711328).