VBNC state and ‘regrowth’ during waste treatment: factors playing a role in loss of culturability of pathogenic bacteria seeded in digestates from agricultural biogas plants

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VBNC state and ‘regrowth’ during waste treatment:

factors playing a role in loss of culturability of pathogenic bacteria seeded in digestates from

agricultural biogas plants

Géraldine Maynaud, Anne-Marie Pourcher, Christine Ziebal, Nathalie Wéry

To cite this version:

Géraldine Maynaud, Anne-Marie Pourcher, Christine Ziebal, Nathalie Wéry. VBNC state and ‘re-

growth’ during waste treatment: factors playing a role in loss of culturability of pathogenic bacteria

seeded in digestates from agricultural biogas plants. HDID V How dead is dead? 5th conference on

exploring the edge of bacterial life, Medical University of Vienna. AUT. Medical University of Graz,

AUT., Sep 2017, Vienne, Austria. �hal-02736308�

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VBNC state and ‘regrowth’ during waste treatment:

factors playing a role in loss of culturability of pathogenic bacteria seeded in digestates from

agricultural biogas plants

Géraldine Maynaud, Anne-Marie Pourcher, Christine Ziebal and Nathalie Wéry

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Comment citer ce document :

Maynaud, G., Pourcher, A.-m., Ziebal, C., Wéry, N. (Présentateur) (2017). VBNC state and

‘regrowth’ during waste treatment: factors playing a role in loss of culturability of pathogenic

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Digestor

Anaerobic digestion

slurry, manure

Food processing waste, fat, sludge from WWTP…

green waste

Digestate

Fate of pathogens?

Protection against microbiological risks

Pathogens

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VBNC state and waste management

Non-culturable Salmonella spp. have shown reactivation potential in composted biosolids with increasing

moisture content and storage (Sidhu et al., 2001)

Reactivation and growth of non-culturable indicator bacteria in anaerobically digested biosolids after centrifuge dewatering

(Higgins et al., 2007; Qi et al., 2007; Viau and Peccia, 2009)

Regrowth during anaerobic mesophilic digestion after thermal pre-treatment - At 55°C, 3h (Pascal-Benito et al., 2015)

- At 70°C, 1h (De los Cobos-Vasconcelos et al., 2015)

Induction of VBNC state of Salmonella sp. and Shigella sp. during thermophilic

anaerobic digestion of biosolids, regain of culturability during storage (Fu et al., 2015)

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Digestor

Objective of the study

slurry, manure

Food processing waste, fat, sludge from WWTP…

green waste

Pathogens STORAGE OF

DIGESTATES

Hygienization

‘Regrowth’

Fate of pathogenic bacteria in stored digestates?

VBNC state induction?

Hygienization

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How to quantify VBNC cells in organic waste?

Look at the gap between culture and qPCR Use qPCR to

quantify viable cells

Membrane integrity assay (PMA-qPCR)

controls with inactivated

inocula

Ayrapetyan, Williams et al. (2015)

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Digestate Nature of sample

Main digester feedstock

Process of AD Phase separation of digestate

Quantity of waste digested per year

(T/year) retention

time (days)

T (°C)

RD1 Raw digestate Pig slurry, fat, green waste 80 42 No 6000

RD2 Raw digestate Cattle slurry and manure, vegetable waste

20-30 37 No 5500

RD3 Raw digestate Cattle manure and slurry 15-16 38 No 12000

LF4 Liquid fraction Pig slurry, food processing waste, maize waste

50-60 38 Drum filter 12500

C7 Compost Pig slurry, biowaste, WWTP sludge, fat

44 38 Centrifugation 12000

LF5 C5

Liquid fraction compost

Manure, green waste 30-32 40 Centrifugation 5000

LF6 C6

Liquid fraction compost

Cattle manure and slurry, poultry manure, green waste

60 41 Screw press 6000

 from agricultural waste (health risk potential)

 Intented for land spreading

 3 raw digestates (RD), 3 liquid fractions (LF), 3 composts (C)

Digestates

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Microcosm assays

Boues1c Boues2b

1. Inoculation of digestates with pathogens (C _i =6-7 log ₁₀ /g):

2. Incubation in conditions simulating storage (24°C for 41 days)

Sampling at T0, 3, 7, 21, 41 days

Salmonella Derby

Listeria monocytogenes

Campylobacter coli

1 ml sampled

3. Quantification of pathogens by culture methods and qPCR

C. coli L. monocytogenes S. derby

Target bacteria and gene

Reference

Salmonella Derby invA Hoorfar et al., 2000

Listeria monocytogenes hlyA Nogva et al., 2000 Campylobacter coli glyA Leblanc-Maridor et

al., 2011

DUPLICATES

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Inactivation curves

RD1 RD2 RD3 LF4 LF5 LF6 C7 C5 C6 RD1 RD2 RD3 LF4 LF5 LF6 C7 C5 C6

Raw digestates

Liquid fraction of digestates

Dried (C7) and composted (C5, C6) solid fraction of digestates

Raw digestates

Higher persistence of Listeria monocytogenes, known to be particularly

resistant to environmental factors (Avery et al., 2014) and rapid inactivation of

Campylobacter coli at 24°C (Bui et al., 2011)

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1. Validation of the controls inoculated with inactivated bacteria 2. VBNC state : LR(qPCR)-LR(culture) > 1 Log

Inactivation curves

RD1 RD2 RD3 LF4 LF5 LF6 C7 C5 RD1 RD2 RD3 LF4 LF5 LF6 C7 C5 C6

Raw digestates

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Pathogenic bacteria in VBNC state in digestates

VBNC state induction of Campylobacter coli, Salmonella Derby and

Listeria monocytogenes after inoculation in digestates was demonstrated but it did not occur in all digestates

Persistence and VBNC induction were digestate-dependent

Validation of all controls apart from C7 which showed low moisture (25%)

preventing degradation of DNA from dead cells

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Salmonella Derby

Listeria

monocytogenes

Campylobacter coli

RD1 RD2 RD3 LF4 LF5 LF6 C5 C6

‘VBNC’

The digestates showing VBNC induction were not the same for the 3 bacteria

Pathogenic bacteria in VBNC state in digestates

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VBNC

Pathogenic bacteria in VBNC state in digestates

Effect of the matrix

Listeria monocytogenes

qPCR culture

Time (d) Time (d)

7d

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Abiotic factors affecting persistence and VBNC induction

NH

₄⁺

/NH

₃

TOC K

₂

O TKN NH

₄⁺

P

₂

O

₅

pH Moisture

%

VM

%

Campylobacter coli

Culture LR - - - 0.6** - - - -

Culture IR - - - - - - - -

qPCR LR - - - -0.5* - - - -

qPCR IR - - - - - - - -

Log C

_qPCR

-Log C

_culture

- - - 0.5* - - - -

Culture LR - - 0.7** - - - -

Culture IR - - 0.6** - - - -

Salmonella Derby qPCR LR - - - - - -0.4*

qPCR IR -0.6 - -0.6 -0.5* - 0.6 -0.6

Culture LR - 0.4* - -0.4* - - -0.5* -

Listeria qPCR LR 0.5* - - - 0.5* - - 0.5*

monocytogenes qPCR IR - - - -0.4* - -0.5* - -

Log C

_qPCR

-Log C

_culture

-0.6** -0.5* - 0.6** -0.5* 0.7* 0.7* -0.6**

LR : Log Removal IR : Inactivation Rate

Pearson's r correlation analysis

low moisture, high nutrient

content,

high VM

COMPOST

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Conclusion

Abiotic factors affecting persistence and VBNC induction

Salmonella Derby : Higher persistence in composted digestates

Listeria monocytogenes : Lower persistence in composted digestates NH ₄ ⁺ /NH ₃ : negative effect on culturability (VBNC state induction)

For L. monocytogenes, effect of pH and moisture on VBNC state induction:

no loss of culturability for digestates showing higher values of pH and moisture

(composts, RD3, LF6)

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Conclusion Conclusion

VBNC state induction of Campylobacter coli, Salmonella Derby and

Listeria monocytogenes after inoculation in digestates was demonstrated Some abiotic factors playing a role in persistence and VBNC induction were identified : NH ₄ ⁺ /NH ₃ , pH, moisture, nutrient content.

Regrowth observed during the storage of digestate, compost or organic waste could be

due to a regain of culturability under favorable conditions after VBNC state induction

Importance of molecular tools such as qPCR for monitoring pathogenic bacteria in

environmental samples since culture methods may underestimate bacterial cell

concentration

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Géraldine Maynaud (Post-doc)

A. Bonnafous N. Wéry

Financially supported by ADEME

(project PRObiotic, n° 1306C0032)

A.-M. Pourcher C. Druilhe

M. Daumoin C. Ziebal Contact : [email protected]

Maynaud, G., Pourcher, A. M., Ziebal, C., Cuny, A., Druilhe, C., Steyer, J. P., & Wéry, N. (2016).

Persistence and potential Viable but Non-culturable state of pathogenic bacteria during storage of

digestates from agricultural biogas plants. Frontiers in Microbiology, 7.