inactivation of pathogenic bacteria in concentrated urine · inactivation of pathogenic bacteria in...

Inactivation of pathogenic bacteriain concentrated urine

Laboratory on Engineering For Sustainable Sanitation,Hokkaido University

Oishi, W., Tezuka, R.,Higikata, N., Ito, R., Ushijima, K., Funamizu, N.

12th Specialized Conference on Small Water and Wastewater System & 4th SpecializedConference on Resources Oriented Sanitation November 2-4, 2014 Muscat, Sultanate of Oman

WHO recommends urine storage at least6 months to avoid infection(WHO,2006)

Pathogenic risk originate fromfecal contamination

INTRODUCTION 2

• Space for urine is limited in urban slum• Storage time should be shortenedBut

More rapid disinfection is necessary

Reuse Urine from Urban slum

http://www.eng.hokudai.ac.jp/labo/UBNWTRSE/outline/index.htm

Urine diverting toilet

UrineFeces Disinfection is necessary

Reuse Urine from Urban slum3

Rural area

Concentration have disinfection potential of pathogen

Volume reduction

Concentration• Volume reduction• Reduce collection and transportation cost• Provide hyperosmolar condition

INTRODUCTION

Objective4

1. Evaluation of relationship between concentration level ofurine and inactivation rate constants of microorganism

2. Examination of predominant factor for inactivation

3. Investigation of inactivation mechanism

Evaluate inactivation of pathogenicbacteria in concentrated urine

INTRODUCTION

Synthetic urine• Non-hydrolyzed and Hydrolyzed• 1~10 folds concentrated

Model microorganism• Escherichia coli (NBRC3301)

Media• Tryptic Soy Agar (TSA)• Desoxycholate Agar (DESO)• Compact Dry EC (C-EC)

MATERIAL AND METHODS 5

TSA DESO C-EC

6

TSA ×

DESO × × ×

C-EC × × ×

DamageonE.coli No specific

damage Enzyme Outermembrane

Enzyme andOuter

membrane

Estimation of damaged parts

MATERIAL AND METHODS

-3.5-3

-2.5-2

-1.5-1

-0.50

0.51

1.5

0 20 40 60

log(N/N0)

Time[hr]

Non-hydrolyzed

1245710

-4

-3.5

-3

-2.5

-2

-1.5

-1

-0.5

00 2 4 6

log(N/N0)

Time[hr]

Hydrolyzed

13510

RESULTS AND DISCUSSION 7

tt kN

dt

dN

dNt/dt：時間tにおける微生物数の変化率，k：不活化速度定数，T-1

Nt：時間tにおける微生物数，t：時間

微生物の不活化を表す式（Ｃｈｉｃｋ，１９０８）

dNt/dt: rate of change in concentration of organisms with time, k:inactivation rate constant, Nt: number of organisms at time t, t: time

( Chick 1908 )

【Evaluation of inactivation rate】

-0.5

0

0.5

1

1.5

2

0 2 4 6 8 10 12

k e[h

-1]

Concentration level

8

Non-hydrolyzed urine

Hydrolyzed urine

• Ke were much higher in hydrolyzed urine• 5 folds concentration was best

Inactivation rate constant in TSA

【Evaluation of inactivation rate】 RESULTS AND DISCUSSION

Working day - exposure frequency -• Scenario 1: 5 times a week (Active collection)• Scenario 2: once a week

9

Collection tank

Risk assessment


1.E-05

1.E-04

1.E-03

1.E-02

1.E-01

1.E+000 2 4 6 8 10

Infe

ctio

us ri

sk[/

yr] Storage time[day]

5 5

1 1

10

Working day[ /week]

Storage time and exposure frequency

Non-hydrolyzedHydrolyzed


• Hydrolyzed urine could shorten required storage time

• Non-hydrolyzed urine require space for storage for 1 week

Examination of the key factor


Inactivation rate was fasterin hydrolyzed urine

What is the factor?

1. NH32. High pH3. Osmotic pressure

-0.5

0

0.5

1

1.5

2

0 5 10 15

k e[h

-1]

Concentration level


Hydrolyzed urine

Condition of Solutions


Synthetic urine Buffer solutions

Non-hydrolyzed

Hydrolyzed AmmoniumSodium

carbonate

NH3 [mM] 0 640-1200 300-1000 0

pH 5.2-5.6 9.2-9.5

Osmotic pressure[mOsm/kg]

754-3220 1233-8300 645-3266 188-8300

Solutions

Parameter

【Examination of the key factor】

-0.4

0

0.4

0.8

1.2

1.6

2

0 500 1000 1500

k e[h

-1]

NH3[mM]

NH₃ buffer

NH₃ buffer

13

NH3 effect【Examination of the key factor】

Difficult to explain inactivation rate constantsby NH3 only

RESULTS AND DISCUSSION

-0.4

0

0.4

0.8

1.2

1.6

2

0 500 1000 1500

k e[h

-1]

NH3[mM]

Hydrolyzed urine


Na₂CO₃ buffer

NH₃ buffer

14

pH effect

There was other predominant factor at high pH

-0.4

0

0.4

0.8

1.2

1.6

2

0 2 4 6 8 10

k e[h

-1]

pH

Hydrolyzed urineNon-hydrolyzed urineNa₂CO₃ bufferNH₃ buffer

【Examination of the key factor】 RESULTS AND DISCUSSION

-0.4

0

0.4

0.8

1.2

1.6

2

0 2000 4000 6000 8000 10000

k e[h

-1]

Osmotic pressure[mOsm/kg]

Hydrolyzed urineNon-hydrolyzed urineNa₂CO₃ bufferNH₃ buffer

15

Osmotic pressure effect

pH9.2-9.5

pH5.2-5.6

Combined effect of osmotic pressure and pHwas the key factor

【Examination of the key factor】 RESULTS AND DISCUSSION

NH3 : 1200mM

Without NH3

0

0.05

0.1

0.15

0.2

0.25

0.3

4 5 7 10

k e[h

-1]

Concentration level

TSA DESO C-EC

16

Damaged part was unclear


【Investigation of inactivation mechanism】

4


17

Hydrolyzed urine

E. coli was damaged on enzyme activity

【Investigation of inactivation mechanism】

0

1

2

3

4

5

6

1 3 5 10

k e[h

-1]

Concentration level

TSA DESO C-EC


CONCLUSIONS 18

1. 5 folds concentration was best for inactivation– In hydrolyzed urine, combined effect of pH and osmotic

pressure caused rapid inactivation

2. Urea hydrolysis reduced required storage time tocollect urine safely– Non-hydrolyzed urine needs space for 1 week storage

3. Enzyme activity was predominantly affected inhydrolyzed urine

Thank you for your attention.

MATERIAL AND METHODS 19

Media CharacterAssumed damages

when result innon-detection

Tryptic Soy Agar(TSA)

Non-selective(bacteria growth media)

Nucleic acid and/orMetabolism

Desoxycholate Agar(DESO)

Selective for E.coli whichhave outer membrane

Membrane and/orNucleic acid and/or

Metabolism

Compact Dry EC(C-EC)

Selective for E.coli whichcan produce beta-

glucuronidase

Enzyme activity and/orNucleic acid and/or

Metabolism

3 media

ScenarioRISK ANALYSIS 20

• Dose-response assessmentModel: Beta-Poisson mode【Ingested pathogen and probability of infection】

P: probability of infection, α,β: parameter, D: number of ingested pathogen

Pinf,year : annual probability of infection, n: frequency of infection

DDP 11)(

n,year P)(P 11inf

ScenarioRISK ANALYSIS 21

• Exposure assessment9.1 mg feces/L urine( Schönning et al., 2002)Infected human feces contain 109 pfu/g feces

– Assumption• 4 member family (infected by rotavirus)• Each of them excretes 1L/day• A collector collects 30 family‘s urine• Urine is concentrated before collected

– Infection route• Urine attach to hand (2.8mL) → oral intake

9.1×103 pfu/Lurine

22

Synthetic urine

N[times] 1 2 3 4 5 6 7 8 9 10 Average

Attached［mL］

2.5 3.0 2.8 3.3 2.5 2.1 2.4 3.3 3.1 3.2 2.8

Result

Exposure: when pour urineUrine attach on collector’s hand.

RISK ANALYSIS

Collection tank

23

Spike E. coli

E.colisolution

…

Tenfold dilution

TSA

DESO

C-EC

Incubate (37oC) Count colony

24h

105-106 cfu/mL-urine

1mL

1mL

1mL

Phosphate buffer 9mL

200mL

Preparesynthetic urine

10 102 ・・・・


24

ReagentsCompounds in each concentration level [mM]

1 2 4 5 7 10MgCl2・6H2O 3.2 6.4 12.8 16 22.4 32

NaCl 78.7 157 315 394 551 787Na2SO4 16.2 32.4 64.8 81 113 162

Na3(C6H5O7)・2H2O 2.6 5.2 10.4 13 18.2 26KCl 21.5 43 86 108 151 215

C4H7N3O 9.7 19.4 38.8 48.5 67.9 97CaCl2・H2O 4.4 8.8 17.6 22 30.8 44

KH2PO4 30.9 61.8 124 155 216 309NH4Cl 18.7 37.4 74.8 93.5 131 187

(NH2)2CO 417 834 1670 2090 2920 4170Na2(COO)2 0.15 0.3 0.6 0.75 1.05 1.5


25

0

1

2

3

4

5

6

1 3 5 10 NaCO₃ buffer

k e[h

-1]

Concentration level of Urine

TSA DESO C-EC


8300mOsm/kg

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