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Induction and persistence of VBNC pathogens during high solid pig manure anaerobic digestion, dewatering, and storage: Biothermal-enhanced vs. conventional systems.

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Τίτλος: Induction and persistence of VBNC pathogens during high solid pig manure anaerobic digestion, dewatering, and storage: Biothermal-enhanced vs. conventional systems.
Συγγραφείς: Zhang J; College of Engineering, China Agricultural University, Beijing 100083, China; Sanya Institute of China Agricultural University, Sanya 572025, China., Westerholm M; Department of Molecular Sciences, Swedish University of Agricultural Sciences, Uppsala 75007, Sweden., Qiao W; College of Engineering, China Agricultural University, Beijing 100083, China; Sanya Institute of China Agricultural University, Sanya 572025, China. Electronic address: qiaowei@cau.edu.cn.
Πηγή: Journal of hazardous materials [J Hazard Mater] 2026 Aug 01; Vol. 514, pp. 142824. Date of Electronic Publication: 2026 Jun 27.
Τύπος έκδοσης: Journal Article
Γλώσσα: English
Στοιχεία περιοδικού: Publisher: Elsevier Country of Publication: Netherlands NLM ID: 9422688 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1873-3336 (Electronic) Linking ISSN: 03043894 NLM ISO Abbreviation: J Hazard Mater Subsets: MEDLINE
Imprint Name(s): Original Publication: Amsterdam : Elsevier
Ιατρικοί όροι (MeSH): Manure*/microbiology , Enterococcus*/growth & development , Enterococcus*/isolation & purification , Salmonella*/growth & development , Salmonella*/isolation & purification , Escherichia coli*/growth & development , Escherichia coli*/isolation & purification, Animals ; Anaerobiosis ; Swine ; Hot Temperature
Περίληψη: Sanitization is a critical foundation for the safe treatment of animal manure. High-temperature anaerobic digestion (AD) is often recommended for pathogen inactivation. However, heat stress may induce microorganisms into a viable-but-non-culturable (VBNC) state rather than truly inactivating them. This study therefore investigated the fate of Escherichia coli, Salmonella, and Enterococcus in pig manure during biothermal hydrolysis-enhanced AD (55 °C followed by 37 °C) and compared it with single-phase mesophilic AD (37 °C). In the two-phase process, culturable E. coli and Salmonella were reduced to below the detection limit (< 1 CFU/g fresh matter) at 55 °C, while Enterococcus achieved a > 5 log10 reduction. Conversely, high levels of culturable E. coli and Enterococcus persisted in the single-phase mesophilic AD. However, propidium monoazide-quantitative PCR revealed substantial VBNC counts (5.6 log10 - 7.8 log10 cells/g fresh matter) in both systems' effluents. Imaging flow cytometry, excitation-emission matrix fluorescence spectroscopy, and ATP measurements further confirmed that these VBNC cells retained membrane integrity and energy metabolism. VBNC cells also showed reduced cell size and amino acid/protein-related signals, indicating a low-metabolic but persistent state. During 129-day storage at ambient temperatures rising from 24 to 30 °C, culturable Salmonella increased to 2 log10-4 log10 CFU/g fresh matter in both digestates while culturable E. coli increased only in the single-phase digestate, likely due to proliferation of residual culturable cells or resuscitation of VBNC cells. These findings reveal that conventional culture-based methods underestimate the actual pathogen load. Optimizing the AD process is therefore necessary to truly eliminate VBNC pathogens for safe land application.
(Copyright © 2026 Elsevier B.V. All rights reserved.)
Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Contributed Indexing: Keywords: Anaerobic digestion; Digestate safety; High solid; Pathogen resuscitation; Pig manure; VBNC pathogens
Substance Nomenclature: 0 (Manure)
Entry Date(s): Date Created: 20260629 Date Completed: 20260725 Latest Revision: 20260725
Update Code: 20260725
DOI: 10.1016/j.jhazmat.2026.142824
PMID: 42372413
Βάση Δεδομένων: MEDLINE
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  Data: Induction and persistence of VBNC pathogens during high solid pig manure anaerobic digestion, dewatering, and storage: Biothermal-enhanced vs. conventional systems.
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  Data: &lt;searchLink fieldCode=&quot;AU&quot; term=&quot;%22Zhang+J%22&quot;&gt;Zhang J&lt;/searchLink&gt;; College of Engineering, China Agricultural University, Beijing 100083, China; Sanya Institute of China Agricultural University, Sanya 572025, China.&lt;br /&gt;&lt;searchLink fieldCode=&quot;AU&quot; term=&quot;%22Westerholm+M%22&quot;&gt;Westerholm M&lt;/searchLink&gt;; Department of Molecular Sciences, Swedish University of Agricultural Sciences, Uppsala 75007, Sweden.&lt;br /&gt;&lt;searchLink fieldCode=&quot;AU&quot; term=&quot;%22Qiao+W%22&quot;&gt;Qiao W&lt;/searchLink&gt;; College of Engineering, China Agricultural University, Beijing 100083, China; Sanya Institute of China Agricultural University, Sanya 572025, China. Electronic address: qiaowei@cau.edu.cn.
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  Data: Sanitization is a critical foundation for the safe treatment of animal manure. High-temperature anaerobic digestion (AD) is often recommended for pathogen inactivation. However, heat stress may induce microorganisms into a viable-but-non-culturable (VBNC) state rather than truly inactivating them. This study therefore investigated the fate of Escherichia coli, Salmonella, and Enterococcus in pig manure during biothermal hydrolysis-enhanced AD (55 &#176;C followed by 37 &#176;C) and compared it with single-phase mesophilic AD (37 &#176;C). In the two-phase process, culturable E. coli and Salmonella were reduced to below the detection limit (&amp;lt; 1 CFU/g fresh matter) at 55 &#176;C, while Enterococcus achieved a &gt; 5 log&lt;subscript&gt;10&lt;/subscript&gt; reduction. Conversely, high levels of culturable E. coli and Enterococcus persisted in the single-phase mesophilic AD. However, propidium monoazide-quantitative PCR revealed substantial VBNC counts (5.6 log&lt;subscript&gt;10&lt;/subscript&gt; - 7.8 log&lt;subscript&gt;10&lt;/subscript&gt; cells/g fresh matter) in both systems&#39; effluents. Imaging flow cytometry, excitation-emission matrix fluorescence spectroscopy, and ATP measurements further confirmed that these VBNC cells retained membrane integrity and energy metabolism. VBNC cells also showed reduced cell size and amino acid/protein-related signals, indicating a low-metabolic but persistent state. During 129-day storage at ambient temperatures rising from 24 to 30 &#176;C, culturable Salmonella increased to 2 log&lt;subscript&gt;10&lt;/subscript&gt;-4 log&lt;subscript&gt;10&lt;/subscript&gt; CFU/g fresh matter in both digestates while culturable E. coli increased only in the single-phase digestate, likely due to proliferation of residual culturable cells or resuscitation of VBNC cells. These findings reveal that conventional culture-based methods underestimate the actual pathogen load. Optimizing the AD process is therefore necessary to truly eliminate VBNC pathogens for safe land application.&lt;br /&gt; (Copyright &#169; 2026 Elsevier B.V. All rights reserved.)
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  Data: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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      – TitleFull: Induction and persistence of VBNC pathogens during high solid pig manure anaerobic digestion, dewatering, and storage: Biothermal-enhanced vs. conventional systems.
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