Journal of Applied Microbiology ISSN 1364-5072
ORIGINAL ARTICLE
Quantification of viable but nonculturable Salmonella spp. and Shigella spp. during sludge anaerobic digestion and their reactivation during cake storage B. Fu1,2,*, Q. Jiang1,2,*, H.-B. Liu1,2 and H. Liu1,2 1 Laboratory of Environmental Biotechnology, School of Environmental and Civil Engineering, Jiangnan University, Wuxi, China 2 Jiangsu Key Laboratory of Anaerobic Biotechnology, Jiangnan University, Wuxi, China
Keywords anaerobic digestion, pathogen, reactivation, sewage sludge, viable but nonculturable. Correspondence He Liu, Laboratory of Environmental Biotechnology, School of Environmental and Civil Engineering, Jiangnan University, Wuxi 214122, China. E-mail: [email protected] *The two authors contributed equally to this article. 2014/2131: received 15 October 2014, revised 1 March 2015 and accepted 24 May 2015 doi:10.1111/jam.12887
Abstract Aims: The presence of viable but nonculturable (VBNC) bacterial pathogens which often fail to be detected by cultivation and can regain the cultivability if the living conditions improve were reported. The objective of this study was to determine the occurrence of VBNC Salmonella spp. and Shigella spp. in the biosolids during anaerobic digestion and its reactivation during the cake storage. Methods and Results: The occurrence of VBNC Salmonella spp. and Shigella spp. during mesophilic, temperature-phased, thermophilic anaerobic digestion of sewage sludge and the subsequent storage were studied by RT-qPCR and most probable number (MPN) method. The VBNC incidence of Salmonella spp. and Shigella spp. during thermophilic digestion was four orders of magnitude higher than those of mesophilic digestion. Accordingly, higher resuscitation ratio of VBNC pathogens was also achieved in thermophilic digested sludge. As a result, the culturable Salmonella typhimurium contents in thermophilic digested sludge after cake storage were two orders of magnitude higher than mesophilic digestion. Both quantitive PCR and reverse transcription quantitative PCR assay results showed the two bacterial counting numbers remained stable throughout the cake storage. Conclusions: The results indicate that the increase in the culturable Salmonella spp. and Shigella spp. after centrifugal dewatering was attributed to the resuscitation from the VBNC state to the culturable state. Significance and Impact of the Study: Thermophilic anaerobic digestion mainly induced Salmonella spp. and Shigella spp. into VBNC state rather than killed them, suggesting that the biological safety of sewage sludge by temperature-phased anaerobic digestion should be carefully assessed.
Introduction As one of the most popular methods for the sustainability of municipal wastewater treatment, anaerobic digestion has been widely applied for the reuse or safe disposal of sewage sludge. Anaerobic digestion brings economic benefits via volatile solids (VS) reduction and biogas production. More importantly, regarding the safety of sludge reuse (such as agricultural application), it has been proven that bacterial pathogens in sewage sludge could be 1138
greatly reduced through anaerobic digestion (Wang et al. 2008). According to the regulations of the US Environmental Protection Agency (USEPA), biosolids are classified as either Class A or Class B biosolids based on pathogen or faecal indicator concentrations and the sludge treatment used (USEPA 1992). The Class A standard was defined as pathogen free (Salmonella spp. below detection levels), whereas Class B biosolids could contain pathogens but can be used. Based on the temperature during the treatments, anaerobic digestion methods
Journal of Applied Microbiology 119, 1138--1147 © 2015 The Society for Applied Microbiology
B. Fu et al.
usually are classified as mesophilic anaerobic digestion (MAD), thermophilic anaerobic digestion (TAD) or temperature-phased anaerobic digestion (TPAD). Class B biosolids treated by MAD are the most prevalent sludge product in the US (Beecher et al. 2007); while TPAD, combining MAD and TAD in separate stages, is one of the most common treatments to obtain Class A biosolids (Godfree and Farrell 2005). In terms of pathogen inactivation, the TAD and TPAD processes are widely recognized to be much more effective than mesophilic digestion (USEPA 1992; Yilmaz et al. 2008). The indicator bacteria or pathogen content in practical evaluation of anaerobic digestion was determined and evaluated by the standard culture methods (SCM) according to the regulations of the USEPA (1992). However, more studies have reported the presence of viable but nonculturable (VBNC) state of indicator bacteria (Higgins et al. 2007; Chen et al. 2011a,b; Erkan and Sanin 2013), which often fail to be detected by cultivation but can regain the cultivability if the living conditions improve. Because of the presence of VBNC indicator bacteria during anaerobic digestion, it is necessary to reassess the inactivation and removal of bacterial pathogens in the biosolids during and after anaerobic digestion. To detect VBNC bacterial pathogen, different methods have been developed to successfully identify the occurrence of VBNC state under different conditions (Viau and Peccia 2009; Liu et al. 2010). However, little is known about the occurrence of VBNC bacterial pathogens during the anaerobic digestion and their fates in the subsequent disposal and land application. Some researchers have applied quantitative PCR (qPCR) method to investigate the VBNC occurrence of indicator bacteria (Higgins et al. 2007; Viau and Peccia 2009; Erkan and Sanin 2013). However, qPCR would overestimate the abundance of viable bacterial cells as DNA from dead cells in digested sludge also could be simultaneously extracted and amplified (Nocker et al. 2006). Alternatively, an increasingly popular method is reverse transcription quantitative PCR (RT-qPCR) targeting at mRNA, which is excellent indicators of bacterial cell viability (Liu et al. 2010). Recently, our group successfully developed a method to quantify the abundance of VBNC bacterial cells in the digested sludge by integrating molecule-based RT-qPCR and cultivation-based most probable number method (MPN) (Jiang et al. 2013). Researchers have reported the content of indicator bacteria in digested sludge immediately increased after centrifugal dewatering and this phenomenon was termed as pathogen ‘regrowth’ (Higgins et al. 2007; Qi et al. 2007; Viau and Peccia 2009; Chen et al. 2011a; Erkan and Sanin 2013). Although pathogen ‘regrowth’ was observed
VBNC pathogens occurrence in sludge
in both thermophilic digestion and mesophilic digestion, this phenomenon was interestingly found more pronounced in thermophilic digested sludge (Higgins et al. 2007; WERF 2008). Based on the previous observations, we hypothesize that (i) thermophilic digestion does not kill the pathogens more efficiently than MAD does, (ii) thermophilic digestion induces more VBNC pathogens to escape from SCM detection and (iii) the increase in pathogen content after dewatering is attributed to the ‘reactivation’ or ‘resuscitation’ of pathogens from the VBNC state. In this study, Salmonella sp. and Shigella sp. were used as model species for assessing the occurrence and reactivation of VBNC bacterial pathogen during different types of anaerobic digestions (MAD, TPAD and TAD). We applied the cultivation-based method MPN and culturefree molecule techniques qPCR/RT-qPCR methods to evaluate the culturable Salmonella sp. and Shigella sp., total Salmonella sp. and Shigella sp. and VBNC Salmonella sp. and Shigella sp. existing in the sludge during MAD, TAD and TPAD digesters and postdigestion cake storage processes. Materials and methods Feed sludge and anaerobic digester operation The sewage sludge (mixed primary sludge and waste activated sludge) collected from Lucun municipal sewage treatment plant (Wuxi, China) was used as feed sludge. The pH of the feed sludge was 68–72 and the total solid (TS) was 5680–6320 g l1. The VS content of TS was 5017–5127% and the VFA, NH4+-N were 6122 and 3233 mg l1 respectively. Before the digestion, the sludge were inoculated with Salmonella enterica serovar Typhimurium and Shigella flexneri, which were kindly provided by Wuxi Center for Disease Control and Prevention (Jiangsu, China). The cells of Salm. typhimurium and Sh. flexneri were grown on LB medium till stationary phase, harvested by centrifugation (8000 g for 5 min), diluted using 5 ml sterile distilled water and then inoculated into the feed sludge with the final concentrations of about 9 log10 MPN g DS1. Five hundred-millilitre flasks containing 300 ml of feed sludge were employed as digesters of sewage sludge, capped and purged with nitrogen. The experiments were carried out in duplicate. The digesters were batch running with appropriate agitation for mixing. The TAD system was operated at 55°C for 26 days and the MAD system was operated at 35°C for 26 days. The TPAD system was operated at thermophilic condition for 3 days, and then at mesophilic conditions for 23 days. The effluent sludge was dewatered centrifugally (10 000 g for
Journal of Applied Microbiology 119, 1138--1147 © 2015 The Society for Applied Microbiology
1139
B. Fu et al.
VBNC pathogens occurrence in sludge
10 min), and then stored at room temperature for the cake storage research. Biogas was released from gas ports and the biogas volume was determined by water discharge method. Chemical analysis methods TS (%), VS (%) and NH4+-N were measured according to the Standard Methods (APHA 2005). For the volatile fatty acid (VFA) measurement, samples were centrifuged and filtered through 045 lm filters. The VFAs concentrations were measured by a gas chromatograph (GC-2010; Shimadzu, Tokyo, Japan) equipped with an auto injector (AOC-20i; Shimadzu). The detector was a flame ionization type and the column was a fused-silica capillary (PEG-20M, 30 m 9 032 mm 9 05 lm, China). Pathogenic analysis Samples were collected every 2 or 3 days during the anaerobic digestion and every 5 days during the dewatered sludge storage for pathogen quantification. Sludge samples were then maintained at 4°C within 6 h for microbial analyses, but both the RNA extraction and MPN enumeration were carried out immediately after sampling. The culturable values of Salmonella sp. and Shigella sp. were quantified by MPN method. The MPN enumeration was carried out following the description of Chen et al. (2012). Five gram of sludge samples were diluted 1 : 10 ranging from 107 to 102 with sterile nutrient broth (for Salmonella spp.) or sterile Gram Negative broth medium (for Shigella spp.). After incubation at 37°C for 8–18 h, 1 ml of the culture was added to 9 ml of Tetrathionate Broth Base medium (for Salmonella spp.) or Shigella-specific broth medium containing novobiocin (for Shigella spp.) in fermentation tubes, which was incubated at 42°C or 37°C for 18–24 h. Samples taken from the fermentation tubes were streaked on Bismuth Sulfite agar plates or Xylose-Lysin-Desoxycholat agar plates using an inoculation loop for Salmonella spp. and Shigella spp., respectively, and incubated at 37°C for 18–24 h. The appearance of black colonies and colourless transparent colonies is a positive reaction that indicates the presence of Salmonella spp. and Shigella spp. respectively. The positive tubes were counted and used to calculate the pathogen density. The detection limit is
ORIGINAL ARTICLE
Quantification of viable but nonculturable Salmonella spp. and Shigella spp. during sludge anaerobic digestion and their reactivation during cake storage B. Fu1,2,*, Q. Jiang1,2,*, H.-B. Liu1,2 and H. Liu1,2 1 Laboratory of Environmental Biotechnology, School of Environmental and Civil Engineering, Jiangnan University, Wuxi, China 2 Jiangsu Key Laboratory of Anaerobic Biotechnology, Jiangnan University, Wuxi, China
Keywords anaerobic digestion, pathogen, reactivation, sewage sludge, viable but nonculturable. Correspondence He Liu, Laboratory of Environmental Biotechnology, School of Environmental and Civil Engineering, Jiangnan University, Wuxi 214122, China. E-mail: [email protected] *The two authors contributed equally to this article. 2014/2131: received 15 October 2014, revised 1 March 2015 and accepted 24 May 2015 doi:10.1111/jam.12887
Abstract Aims: The presence of viable but nonculturable (VBNC) bacterial pathogens which often fail to be detected by cultivation and can regain the cultivability if the living conditions improve were reported. The objective of this study was to determine the occurrence of VBNC Salmonella spp. and Shigella spp. in the biosolids during anaerobic digestion and its reactivation during the cake storage. Methods and Results: The occurrence of VBNC Salmonella spp. and Shigella spp. during mesophilic, temperature-phased, thermophilic anaerobic digestion of sewage sludge and the subsequent storage were studied by RT-qPCR and most probable number (MPN) method. The VBNC incidence of Salmonella spp. and Shigella spp. during thermophilic digestion was four orders of magnitude higher than those of mesophilic digestion. Accordingly, higher resuscitation ratio of VBNC pathogens was also achieved in thermophilic digested sludge. As a result, the culturable Salmonella typhimurium contents in thermophilic digested sludge after cake storage were two orders of magnitude higher than mesophilic digestion. Both quantitive PCR and reverse transcription quantitative PCR assay results showed the two bacterial counting numbers remained stable throughout the cake storage. Conclusions: The results indicate that the increase in the culturable Salmonella spp. and Shigella spp. after centrifugal dewatering was attributed to the resuscitation from the VBNC state to the culturable state. Significance and Impact of the Study: Thermophilic anaerobic digestion mainly induced Salmonella spp. and Shigella spp. into VBNC state rather than killed them, suggesting that the biological safety of sewage sludge by temperature-phased anaerobic digestion should be carefully assessed.
Introduction As one of the most popular methods for the sustainability of municipal wastewater treatment, anaerobic digestion has been widely applied for the reuse or safe disposal of sewage sludge. Anaerobic digestion brings economic benefits via volatile solids (VS) reduction and biogas production. More importantly, regarding the safety of sludge reuse (such as agricultural application), it has been proven that bacterial pathogens in sewage sludge could be 1138
greatly reduced through anaerobic digestion (Wang et al. 2008). According to the regulations of the US Environmental Protection Agency (USEPA), biosolids are classified as either Class A or Class B biosolids based on pathogen or faecal indicator concentrations and the sludge treatment used (USEPA 1992). The Class A standard was defined as pathogen free (Salmonella spp. below detection levels), whereas Class B biosolids could contain pathogens but can be used. Based on the temperature during the treatments, anaerobic digestion methods
Journal of Applied Microbiology 119, 1138--1147 © 2015 The Society for Applied Microbiology
B. Fu et al.
usually are classified as mesophilic anaerobic digestion (MAD), thermophilic anaerobic digestion (TAD) or temperature-phased anaerobic digestion (TPAD). Class B biosolids treated by MAD are the most prevalent sludge product in the US (Beecher et al. 2007); while TPAD, combining MAD and TAD in separate stages, is one of the most common treatments to obtain Class A biosolids (Godfree and Farrell 2005). In terms of pathogen inactivation, the TAD and TPAD processes are widely recognized to be much more effective than mesophilic digestion (USEPA 1992; Yilmaz et al. 2008). The indicator bacteria or pathogen content in practical evaluation of anaerobic digestion was determined and evaluated by the standard culture methods (SCM) according to the regulations of the USEPA (1992). However, more studies have reported the presence of viable but nonculturable (VBNC) state of indicator bacteria (Higgins et al. 2007; Chen et al. 2011a,b; Erkan and Sanin 2013), which often fail to be detected by cultivation but can regain the cultivability if the living conditions improve. Because of the presence of VBNC indicator bacteria during anaerobic digestion, it is necessary to reassess the inactivation and removal of bacterial pathogens in the biosolids during and after anaerobic digestion. To detect VBNC bacterial pathogen, different methods have been developed to successfully identify the occurrence of VBNC state under different conditions (Viau and Peccia 2009; Liu et al. 2010). However, little is known about the occurrence of VBNC bacterial pathogens during the anaerobic digestion and their fates in the subsequent disposal and land application. Some researchers have applied quantitative PCR (qPCR) method to investigate the VBNC occurrence of indicator bacteria (Higgins et al. 2007; Viau and Peccia 2009; Erkan and Sanin 2013). However, qPCR would overestimate the abundance of viable bacterial cells as DNA from dead cells in digested sludge also could be simultaneously extracted and amplified (Nocker et al. 2006). Alternatively, an increasingly popular method is reverse transcription quantitative PCR (RT-qPCR) targeting at mRNA, which is excellent indicators of bacterial cell viability (Liu et al. 2010). Recently, our group successfully developed a method to quantify the abundance of VBNC bacterial cells in the digested sludge by integrating molecule-based RT-qPCR and cultivation-based most probable number method (MPN) (Jiang et al. 2013). Researchers have reported the content of indicator bacteria in digested sludge immediately increased after centrifugal dewatering and this phenomenon was termed as pathogen ‘regrowth’ (Higgins et al. 2007; Qi et al. 2007; Viau and Peccia 2009; Chen et al. 2011a; Erkan and Sanin 2013). Although pathogen ‘regrowth’ was observed
VBNC pathogens occurrence in sludge
in both thermophilic digestion and mesophilic digestion, this phenomenon was interestingly found more pronounced in thermophilic digested sludge (Higgins et al. 2007; WERF 2008). Based on the previous observations, we hypothesize that (i) thermophilic digestion does not kill the pathogens more efficiently than MAD does, (ii) thermophilic digestion induces more VBNC pathogens to escape from SCM detection and (iii) the increase in pathogen content after dewatering is attributed to the ‘reactivation’ or ‘resuscitation’ of pathogens from the VBNC state. In this study, Salmonella sp. and Shigella sp. were used as model species for assessing the occurrence and reactivation of VBNC bacterial pathogen during different types of anaerobic digestions (MAD, TPAD and TAD). We applied the cultivation-based method MPN and culturefree molecule techniques qPCR/RT-qPCR methods to evaluate the culturable Salmonella sp. and Shigella sp., total Salmonella sp. and Shigella sp. and VBNC Salmonella sp. and Shigella sp. existing in the sludge during MAD, TAD and TPAD digesters and postdigestion cake storage processes. Materials and methods Feed sludge and anaerobic digester operation The sewage sludge (mixed primary sludge and waste activated sludge) collected from Lucun municipal sewage treatment plant (Wuxi, China) was used as feed sludge. The pH of the feed sludge was 68–72 and the total solid (TS) was 5680–6320 g l1. The VS content of TS was 5017–5127% and the VFA, NH4+-N were 6122 and 3233 mg l1 respectively. Before the digestion, the sludge were inoculated with Salmonella enterica serovar Typhimurium and Shigella flexneri, which were kindly provided by Wuxi Center for Disease Control and Prevention (Jiangsu, China). The cells of Salm. typhimurium and Sh. flexneri were grown on LB medium till stationary phase, harvested by centrifugation (8000 g for 5 min), diluted using 5 ml sterile distilled water and then inoculated into the feed sludge with the final concentrations of about 9 log10 MPN g DS1. Five hundred-millilitre flasks containing 300 ml of feed sludge were employed as digesters of sewage sludge, capped and purged with nitrogen. The experiments were carried out in duplicate. The digesters were batch running with appropriate agitation for mixing. The TAD system was operated at 55°C for 26 days and the MAD system was operated at 35°C for 26 days. The TPAD system was operated at thermophilic condition for 3 days, and then at mesophilic conditions for 23 days. The effluent sludge was dewatered centrifugally (10 000 g for
Journal of Applied Microbiology 119, 1138--1147 © 2015 The Society for Applied Microbiology
1139
B. Fu et al.
VBNC pathogens occurrence in sludge
10 min), and then stored at room temperature for the cake storage research. Biogas was released from gas ports and the biogas volume was determined by water discharge method. Chemical analysis methods TS (%), VS (%) and NH4+-N were measured according to the Standard Methods (APHA 2005). For the volatile fatty acid (VFA) measurement, samples were centrifuged and filtered through 045 lm filters. The VFAs concentrations were measured by a gas chromatograph (GC-2010; Shimadzu, Tokyo, Japan) equipped with an auto injector (AOC-20i; Shimadzu). The detector was a flame ionization type and the column was a fused-silica capillary (PEG-20M, 30 m 9 032 mm 9 05 lm, China). Pathogenic analysis Samples were collected every 2 or 3 days during the anaerobic digestion and every 5 days during the dewatered sludge storage for pathogen quantification. Sludge samples were then maintained at 4°C within 6 h for microbial analyses, but both the RNA extraction and MPN enumeration were carried out immediately after sampling. The culturable values of Salmonella sp. and Shigella sp. were quantified by MPN method. The MPN enumeration was carried out following the description of Chen et al. (2012). Five gram of sludge samples were diluted 1 : 10 ranging from 107 to 102 with sterile nutrient broth (for Salmonella spp.) or sterile Gram Negative broth medium (for Shigella spp.). After incubation at 37°C for 8–18 h, 1 ml of the culture was added to 9 ml of Tetrathionate Broth Base medium (for Salmonella spp.) or Shigella-specific broth medium containing novobiocin (for Shigella spp.) in fermentation tubes, which was incubated at 42°C or 37°C for 18–24 h. Samples taken from the fermentation tubes were streaked on Bismuth Sulfite agar plates or Xylose-Lysin-Desoxycholat agar plates using an inoculation loop for Salmonella spp. and Shigella spp., respectively, and incubated at 37°C for 18–24 h. The appearance of black colonies and colourless transparent colonies is a positive reaction that indicates the presence of Salmonella spp. and Shigella spp. respectively. The positive tubes were counted and used to calculate the pathogen density. The detection limit is
