Inactivation mechanism of pathogenic
bacteria using lime and ash
in composting toilet
★Rui TEZUKA,
Nowaki HIJIKATA, Shinobu KAZAMA, Seyram K. SOSSOU, Naoyuki FUNAMIZU
Department of Environmental Engineering, Hokkaido University
2 Background
Composting toilet
Low initial / running cost
No electric energy needs
Utilization of finished compost as fertilizer
It is effective to improve the sanitary condition
in developing countries.
• Compost has a potential to trap pathogens.
• Exchange of matrix involves high infection risk.
Some sanitary treatment must be applied.
Introduction
http://www.idahostatesman.com/2012/06/28/2171123/composting-is-not-difficult-but.html
3
Methods for pathogen reduction
Chlorine (Oxidizer)
UV radiation
Drying
Heating with sunlight
Alkaline treatment
Calcium lime / ash
(Gunter et al., 2005)
(Kasama et al., 2010)
←At low cost
←Composting Toilet
The information of alkaline treatment is still limited.
Sanitary treatment
However
Introduction
4 Objective
Big Objective
To develop the production of safe compost by alkaline treatment
Relationship between applied CaO / ash amount and compost pH
Alkalinity level and inactivation rate
Damage in pathogenic bacteria
Required amount of CaO to ruduce infection risk (from the viewpoint of risk assessment)
To approach this objective…
5 Experimental conditions
Matrix （Initial：20[L]) Charcoal
(Rice husk)
Rice husk
Composting period （Input feces:500[g/day]）
About 1 month (38 days) About 1 month (38 days)
Fecal degradation rate 41.2% 45.8%
Water content About 50 % (adjusted)
Experimental temperature 37℃(mesosphiric)
Indicator Escherichia coli NBRC3301
Alkaline material •Calcium Oxide (as calcium lime, Wako chemical)
•Wood ash (Maruta Soumokubai K.100)
Compost conditions
Experimental conditions
Materials and Methods
6 3 media
Media Characteristic Damage parts of E. coli
TSA Non-selective Nucleic acid and/or metabolism
DESO Selective for gram negative
(gram positive can’t grow）
Outer membrane and/or nucleic
acid and/or metabolism
C-EC Selective for bacteria which can
select b-glucuronidase
Enzyme Activity and/or nucleic
acid and/or metabolism
TSA (Tryptic Soy Agar)
DESO (Desooxicholate Agar)
C-EC (Compact Dry - EC) TSA
DESO
C-EC
Materials and Methods
7 Experimental procedure
E. Coli
Extraction of
E. coli Dilution
media
Sterilized
feces
CaO / ash
Measurement
Inoculation in media
E. Coli is prepared
Sterilized
compost
Materials and Methods
8
9
10
11
12
0 1 2 3 4 5
Applied CaO / ash amount per 1 g-DW of compost [g/g-DW]
pH
CaO (Charcoal)CaO (Rice husk)Wood ash (Rice husk)
8 The pH of Compost
Based on this relationship, compost pH was adjusted.
(without adjustment, pH 10.0, 10.5)
CaO can increase the compost pH more rapidly than ash.
Compost 5[g]
+ Water 100[mL]
+ CaO/Wood Ash
→Shaking 30 min
→ pH meter
pH measurement
8
9
10
11
12
0 5 10 15 20 25 30
Applied CaO / ash amount per 1 g-DW of compost [mg/g-DW]
pH
CaO (Charcoal)CaO (Rice husk)Wood ash (Rice husk)
Results and Discussion
9 Concentration of E. coli
Inactivation of microorganism (Nakagawa et al., 2006, Otaki et al., 2007)
N,N0: concentration of microorganisms at time t and at time 0 k: inactivation rate constant(IRC) t: retention time
Based on these plots , IRC / Normalized IRC were calculated
-5
-4
-3
-2
-1
0
0 2 4 6 8Time [h]
log(N
/N0)
TSA (without CaO) DESO (without CaO) C-EC (without CaO)
TSA (pH=10.1) DESO (pH=10.1) C-EC (pH=10.1)
TSA (pH=10.5) DESO (pH=10.5) C-EC (pH=10.5)
TSA (pH=10.0) (wood ash) DESO (pH=10.0) (wood ash) C-EC (pH=10.0) (wood ash)
-5
-4
-3
-2
-1
0
0 2 4 6 8Time [h]
log
(N/N
0)
Charcoal Rice husk
Results and Discussion
10 Normalized IRC
0
0.5
1
1.5
2
2.5
pH 9.6
(without CaO)
pH 10.1 pH 10.4 pH 9.1
(without CaO)
pH 10.1 pH 10.5 pH 10.0
Charcoal (CaO) Rice husk (CaO) Rice husk
(Wood Ash)
No
rmali
zed
in
acti
vati
on
rate
co
nst
an
t [h
-1
]
Normalized IRC increased as pH increases.
Charcoal was more likely to be affected by alkalization.
Wood ash was more efficient than CaO at the same pH level.
×8
×12
×2
×4 ×4
Results and Discussion
11 IRC on each media
Charcoal： Outer membrane and enzyme activity
Rice husk： enzyme activity
CaO / Ash：Similar tendency
：Nucleic / metabolism
：Outer membrane
：Enzyme activity
Results and Discussion
12 Risk assessment
Infection Scenario
Four member family (infected by Salmonella), 150g of feces [ /person / day]
Matrix is exchanged every 3 months (Compost weight: about 40 [L] (=30 [kg-DW]))
Before exchange the matrix, CaO is applied.
Oral intake at a time is 500 [mg] (Nakata et al., 2003)
Annual acceptable level：10-4[/yr] ⇒ 3.2×10-5[/a time]
P(D) ：acceptable level of infection [infection/person/year]
P:acceptable probability of [infection/person/a time] n：number of exposure event
D：exposure dose a,b：model parameter (Salmonella: a = 0.33, b = 139.9)
a
b
DDP 11)(
nP)1(1infection of level Acceptable
Infection risk ：beta-poisson model
Materials and Methods
13
1.E-07
1.E-06
1.E-05
1.E-04
1.E-03
1.E-02
1.E-01
1.E+00
0 2 4 6 8 10 12 14 16 18 20 22 24
Time[h]
Infe
ctio
n R
isk
without CaOpH 10.1pH 10.2pH 10.3pH 10.4pH 10.5
Charcoal case
Goal：To reduce the infection risk to 3.2 × 10-5 within 4 hours
Infection risk (Charcoal)
pH 10.2 (CaO input：27[mg/g-compost (dry base)]
On assumed toilet：810[g] (compost ：30[kg-DW])
Results and Discussion
14
1.E-07
1.E-06
1.E-05
1.E-04
1.E-03
1.E-02
1.E-01
1.E+00
0 2 4 6 8 10 12 14 16 18 20 22 24
Time[h]
Infe
ctio
n R
isk
without CaO
pH 10.1pH 10.2
pH 10.3pH 10.4
pH 10.5
Infection risk (Rice husk)
Rice husk case
Goal：To reduce the infection risk to 3.2 × 10-5 within 4 hours
pH 10.2 (CaO input：28[mg/g-compost (dry base)]
On assumed toilet：840[g] (compost ：30[kg-DW])
Results and Discussion
15 Conclusions
1. To decrease infection risk, about 900 g of CaO was required. (On the assumption that four member family use the composting toilet for 3
months)
2. High pH tended to damage outer membrane and enzyme
activity to pathogenic bacteria.
3. At the same pH level, wood ash tended to damage more
lethally than CaO but larger amount was required.
16
17
Composting
toilet
Farmland
(For Agriculture)
feces grey water
Grey water
Treatment
Onsite Wastewater Differentiable Treatment System
urine
OWDTS
18
Amount of inputted CaO
Compost made from sludge(Sapporo Compost*)：18%
Inputted CaO in this experiment：3% at most
Amount of applied wood ash
To raise the compost pH to 10, 10g of wood ash was applied to 20 g of compost. ：50% at mass ratio →Too much ??
Contribution of pH on plant
Dilution by the soil
Buffering by organic acid or decaying vegetable matter
*：http://www.city.sapporo.jp/gesui/04chishiki/03conpo.html
Photo：http://www.soil-doctor.jp/material/am/
Proper amount ??
Effect on plant growth
19
Changeable factors by autoclave
Volatile growth inhibitor (=change in chemical characterization)
Other microorganisms (= competition) (Pietronave et al., 2004)
Estimated death/inactivation causes for microorganism
Temperature （= Thermal inactivation)
Water content （=drying）
pH (high / low pH)
Nutrient conditons (Nitrogen, Phosphorus)
Effect on autoclaved sterilization
More significant
Controllable
pH increase by CaO and Ash (dissoleved in pure water)
6
7
8
9
10
11
12
0 1 2 3 4 5
[g/mL-pure water]
pH
Ash CaO
20 Alkalinity level of CaO / wood ash
◆：Wood ash
■：CaO
pH measurement
Pure water
CaO/ ash
pH increase by CaO and Ash (dissoleved in pure water)
6
7
8
9
10
11
12
0 0.02 0.04 0.06 0.08 0.1
[g/mL-pure water]
pH
CaO Ash
In the case of wood ash, the pH increase was slighter than CaO.
To increase the same pH level, larger amount of ash is required.
21
Tar (contained in charcoal)
Antibacterial substance such as phenolic acid, lignin (Charcoal < Rice husk)
Inorganic matter (Charcoal > Rice husk)
pH increase pattern
Degree of inactivation
Difference in each matrixes
Relation??
Characteristic of wood ash
Maruta Soumokubai K.100
pH 12.3
Total content of phosphate :20.7 %
Total K:24.0%
Compost 5[g]
+ Water 100[mL]
+ Wood Ash
Shaking for 30 minites pH
measurement
pH measurement / wood ash
Compost pH measurement
23
Assumed type of composting toilet
Matrix：about 40[L]
Capacity：96[L]
Cost：10,000 yen
Compost weight： about 30[kg-DW]
(after 3 months working)
Photo above one by K.Yabui,
below one is from http://www.eng.hokudai.ac.jp/labo/UBNWTRSE/outline/index.htm
（Yabui et al., 2010）
Type of composting toilet
