Inactivation of pathogenic bacteria in concentrated urine

Transcription

1 12 th Specialized Conference on Small Water and Wastewater System & 4 th Specialized Conference on Resources Oriented Sanitation November 2-4, 2014 Muscat, Sultanate of Oman Inactivation of pathogenic bacteria in concentrated urine Oishi, W., Tezuka, R., Higikata, N., Ito, R., Ushijima, K., Funamizu, N. Laboratory on Engineering For Sustainable Sanitation, Hokkaido University

2 Reuse Urine from Urban slum INTRODUCTION 2 Urine diverting toilet Pathogenic risk originate from fecal contamination Feces Urine Disinfection is necessary WHO recommends urine storage at least 6 months to avoid infection(who,2006) But Space for urine is limited in urban slum Storage time should be shortened More rapid disinfection is necessary

3 Reuse Urine from Urban slum Volume reduction INTRODUCTION 3 Rural area Concentration Volume reduction Reduce collection and transportation cost Provide hyperosmolar condition Concentration have disinfection potential of pathogen

4 Objective INTRODUCTION 4 Evaluate inactivation of pathogenic bacteria in concentrated urine 1. Evaluation of relationship between concentration level of urine and inactivation rate constants of microorganism 2. Examination of predominant factor for inactivation 3. Investigation of inactivation mechanism

5 Synthetic urine Non-hydrolyzed and Hydrolyzed 1~10 folds concentrated MATERIAL AND METHODS 5 Model microorganism Escherichia coli (NBRC3301) Media Tryptic Soy Agar (TSA) Desoxycholate Agar (DESO) Compact Dry EC (C-EC) TSA DESO C-EC

6 MATERIAL AND METHODS 6 Estimation of damaged parts Damage on E.coli No specific damage Enzyme Outer membrane Enzyme and Outer membrane TSA DESO C-EC

7 Evaluation of inactivation rate RESULTS AND DISCUSSION 7 log(n/n 0 ) Hydrolyzed Time[hr] dnt dt log(n/n 0 ) kn t Non-hydrolyzed ( Chick 1908 ) Time[hr] dn t /dt: rate of change in concentration of organisms with time, k: inactivation rate constant, N t : number of organisms at time t, t: time

8 Evaluation of inactivation rate RESULTS AND DISCUSSION Inactivation rate constant in TSA 8 2 Hydrolyzed urine k e [h -1 ] Non-hydrolyzed urine Concentration level K e were much higher in hydrolyzed urine 5 folds concentration was best

9 Evaluation of inactivation rate RESULTS AND DISCUSSION 9 Risk assessment Working day - exposure frequency - Scenario 1: 5 times a week (Active collection) Scenario 2: once a week Collection tank

10 Evaluation of inactivation rate RESULTS AND DISCUSSION Storage time and exposure frequency 10 Infectious risk[/yr] 1.E+00 1.E-01 1.E-02 1.E-03 1.E Storage time[day] Hydrolyzed Non-hydrolyzed Working day[ /week] Hydrolyzed 1.E-05 urine could shorten required storage time Non-hydrolyzed urine require space for storage for 1 week

11 Examination of the key factor RESULTS AND DISCUSSION 11 k e [h -1 ] Hydrolyzed urine Non-hydrolyzed urine Inactivation rate was faster in hydrolyzed urine What is the factor? Concentration level 1. NH 3 2. High ph 3. Osmotic pressure

12 Examination of the key factor RESULTS AND DISCUSSION 12 Condition of Solutions Solutions Synthetic urine Buffer solutions Parameter Nonhydrolyzed Hydrolyzed Ammonium Sodium carbonate NH 3 [mm] ph Osmotic pressure [mosm/kg]

13 Examination of the key factor NH 3 effect 2 RESULTS AND DISCUSSION 13 k e [h -1 ] NH NH 3 [mm] 3 [mm] Hydrolyzed urine Non-hydrolyzed urine Na₂CO₃ buffer NH₃ buffer NH₃ buffer Difficult to explain inactivation rate constants by NH 3 only

14 Examination of the key factor RESULTS AND DISCUSSION ph effect 14 k e [h -1 ] Hydrolyzed urine Non-hydrolyzed urine Na₂CO₃ buffer NH₃ buffer There was other predominant factor at high ph ph

15 Examination of the key factor RESULTS AND DISCUSSION Osmotic pressure effect 2 NH 3 : 1200mM 15 k e [h -1 ] ph ph Osmotic pressure[mosm/kg] Hydrolyzed urine Non-hydrolyzed urine Na₂CO₃ buffer NH₃ buffer Combined effect of osmotic pressure and ph was the key factor Without NH 3

16 Investigation of inactivation mechanism Non-hydrolyzed urine RESULTS AND DISCUSSION TSA DESO C-EC 16 k e [h -1 ] Concentration level Damaged part was unclear

17 Investigation of inactivation mechanism Hydrolyzed urine 6 5 TSA DESO C-EC RESULTS AND DISCUSSION 17 k e [h -1 ] Concentration level E. coli was damaged on enzyme activity

18 CONCLUSIONS 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 to collect urine safely Non-hydrolyzed urine needs space for 1 week storage 3. Enzyme activity was predominantly affected in hydrolyzed urine Thank you for your attention.

19 3 media Media Tryptic Soy Agar (TSA) Desoxycholate Agar (DESO) Character Non-selective (bacteria growth media) Selective for E.coli which have outer membrane MATERIAL AND METHODS 19 Assumed damages when result in non-detection Nucleic acid and/or Metabolism Membrane and/or Nucleic acid and/or Metabolism Compact Dry EC (C-EC) Selective for E.coli which can produce betaglucuronidase Enzyme activity and/or Nucleic acid and/or Metabolism

20 Scenario Dose-response assessment Model: Beta-Poisson mode Ingested pathogen and probability of infection P( D) 1 1 D RISK ANALYSIS 20 P: probability of infection, α,β: parameter, D: number of ingested pathogen P inf n,year 1 ( 1 P) P inf,year : annual probability of infection, n: frequency of infection

21 RISK ANALYSIS 21 Scenario Exposure assessment 9.1 mg feces /L urine ( Schönning et al., 2002) Infected human feces contain 10 9 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 pfu/l urine

22 RISK ANALYSIS 22 Synthetic urine Exposure: when pour urine Urine attach on collector s hand. Collection tank N[times] Average Attached [ml] Result

23 MATERIAL AND METHODS mL Spike E. coli cfu/ml -urine Prepare synthetic urine E.coli solution Phosphate buffer 9mL Tenfold dilution 1mL TSA Incubate (37 o C) Count colony 1mL 1mL DESO C-EC 24h

24 MATERIAL AND METHODS 24 Reagents Compounds in each concentration level [mm] MgCl 2 6H 2 O NaCl Na 2 SO Na 3 (C 6 H 5 O 7 ) 2H 2 O KCl C 4 H 7 N 3 O CaCl 2 H 2 O KH 2 PO NH 4 Cl (NH 2 ) 2 CO Na 2 (COO)

25 RESULTS AND DISCUSSION TSA DESO C-EC 8300mOsm/kg k e [h -1 ] NaCO₃ buffer Concentration level of Urine