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CRITICAL REVIEW

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Technol., 2023, 9, 1008

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Resource recovery technologies as microbial risk
barriers: towards safe use of excreta in agriculture
based on hazard analysis and critical control
point†
Wakana Oishi,*a Björn Vinneråsb and Daisuke Sanoac

Agricultural use of human excreta contributes to sustainable nutrient resource management. In contrast,
resource recovery from human excreta is associated with the risk of infection by pathogenic
microorganisms. The microbial risk associated with human excreta needs to be properly managed.
Pathogen inactivation efficacy of resource recovery technologies should be evaluated so that individual
resource recovery processes can be monitored as health risk barriers. To this end, we reviewed thesanitization potential of resource recovery technologies from human excreta and identified the monitoring
parameters of hazard analysis and critical control point (HACCP). We describe the inactivation of surrogate
microorganisms in selected technologies in terms of the physicochemical conditions of matrices, different
tolerances among surrogate microorganisms, and inactivation mechanisms. The estimated storage/
operating time required to achieve the target log reduction values (LRVs) is shortened in thermal processes
such as thermophilic storage, drying, composting, microwaving, and thermophilic digestion. In mostReceived 1st November 2022,
Accepted 26th February 2023
DOI: 10.1039/d2ew00832g

processes, phage and helminth eggs were found to be appropriate indicators for conservative estimation.
Finally, we argue that sanitization is congruent with decomposition of readily degradable organic matterand resource recovery. The barrier efficacy of resource recovery technologies should be validated using apathogen inactivation kinetics model so that the exposure risk to infectious pathogens is sufficiently

rsc.li/es-water

reduced through a reasonable combination of non-treatment and non-technical health risk barriers.

Water impact
Pathogens in human excreta can be inactivated through physical, chemical, and biological processes for recovering plant nutrients and organics in humanexcreta. This study summarizes the sanitization potential and important factors that affect the inactivation rate of pathogens in excreta matrices, aiding theidentification of appropriate operational parameters to be monitored to improve microbiological quality of the final products.

1. Introduction
Nutrient recycling of human excreta for agricultural
production is a sustainable nutrient management approachto feed the growing global population.1 A paradigm shift thatreframes sanitation as part of integrated nutrienta

Department of Civil and Environmental Engineering, Graduate School ofEngineering, Tohoku University, Aoba 6-6-06, Aramaki, Aoba-ku, Sendai, Miyagi
980-8597, Japan. E-mail: wakana.oishi.d1@tohoku.ac.jp; Tel: +81 22 795 3584b
Department of Energy and Technology, Swedish University of Agricultural
Sciences, Box 7032, 75007, Uppsala, Swedenc
Department of Frontier Sciences for Advanced Environment, Graduate School ofEnvironmental Studies, Tohoku University, Aoba 6-6-06, Aramaki, Aoba-ku, Sendai,
Miyagi 980-8597, Japan† Electronic supplementary information (ESI) available. See DOI: https://doi.org/
10.1039/d2ew00832g

1008 | Environ. Sci.: Water Res. Technol., 2023, 9, 1008–1029

management in farming systems was proposed by Harder
et al.2 Human excreta are particularly rich in plant nutrientsas an average person excretes 4.5 kg of nitrogen, 0.5 kg ofphosphorus, and 1.2 kg of potassium every year.3 Thesenutrients excreted from the global populations amount to 41
million tones, which represents 28% of the current
consumption of nitrogen, phosphorus, and potassium.4
Agricultural resources are recovered from human excreta
using appropriate sanitation technologies.5 However, humanexcreta contain pathogens that cause infectious diseases. To
protect public health, resource recovery activities should be
guided by an appropriate health risk management program.
Additional benefits from treating and recycling the excreta isthat we decrease the environmental pollution and stop
pathogens from reaching the environment.

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Environmental Science: Water Research & Technology
The WHO Guidelines6 provide a comprehensive
framework for health risk management of agricultural use ofhuman excreta. The framework consists of 1) identificationof risk points, 2) identification of parameters for monitoringrisk points, 3) development of multiple barriers, 4)monitoring strategy for developed barriers, and 5) validationof the effectiveness of health protection measures.6
Sanitation Safety Planning (SSP) is a tool that guides local
authorities and practitioners to implement the 2006 WHO
guidelines.
SSP uses the methods and procedures of hazard analysis
and critical control point (HACCP) in the operational
monitoring and validation of a sanitation system.7 The
fundamental principle of HACCP is monitoring operationalparameters (hereinafter monitoring parameters) and record
keeping at a critical control point (CCP), which is a vital
process to reduce infection risk. The critical limits (CLs) of
the monitoring parameter are defined as the standard values
that must be achieved at the CCPs.8 Technological microbial
risk barriers, such as storage and treatment processes, aremonitored as CCPs in sanitation systems (Fig. 1). Microbial

Wakana Oishi

Björn Vinnerås

Dr. Wakana Oishi is an assistantprofessor in the Department of
Civil
and
Environmental
Engineering
atTohokuUniversity. Her research focuseson microbial risk management
for the safe resource recovery
from human excreta. She
specializes in the modeling ofinactivation of pathogens inenvironmental matrices andinfection risk associated withrecycling human excreta.

Dr. Björn Vinnerås is a professor
in the Department of Energy andTechnology at the Swedish
UniversityofAgricultural
Sciences. He specializes indeveloping technologies to recycle
plant nutrients from humanexcreta back to agricultural fields
in an efficient and safe way, andmainly works on hygiene
technology, protein production,and recovering plant nutrientsfrom source separating sewage
systems.

Critical review

Fig. 1 Implementation of the HACCP approach to a process train forresource recovery from human excreta. A process train is composedof sequential treatment units, including sedimentation, dewatering,stabilization of readily degradable organic matter, and storage. A
critical control point (CCP) identified among the continuous processes
is monitored with monitoring parameters so that they meet the criticallimits (CLs).

concentration is usually not used as a monitoring parameter
for CCPs. Chemical and physical indicators that reflect thelevel of pathogens and can be quickly monitored are moresuitable monitoring parameters; thus, the pathogen
concentration in products from sanitation systems isindirectly estimated based on the monitoring parameters,
which is the basis of HACCP. The CLs can be determined inadvance using a mathematical model that is a function ofchemical and physical indicators (Fig. 1).9
When pathogens are inactivated in a resource recovery
process, the process functions as a health risk barrier andshould be monitored as CCP. The WHO recommends
operational monitoring of indicators that are quickly
monitored but represent the pathogen level; it recommends
storage time based on the temperature and pH of fecal
sludge.6 Meanwhile, for emerging resource recovery systems
and practices which are not restricted by the WHO
guidelines, it recommends voluntary evaluation of pathogen
inactivation values and validation of the microbial quality ofDaisuke Sano

Dr. Daisuke Sano is a professor
in the Department of Civil andEnvironmental Engineering at
Tohoku University. He specializes
in health-related water virology
and public health. His group's
research has focused on waterand public health issues,
including anti-microbial resistantbacteria in a water environment,
histo-bloodgroup
antigenpositive bacteria in the ecology ofhumannoroviruses,
andmodeling of enteric viruses'

disinfection/removal.

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Environmental Science: Water Research & Technology

products from sanitation systems.6 The validation thresholds
are specified by the WHO, International Organization forStandardization (ISO), and local governments.6,10–13 The ISO
30500 specifies the target log reduction values of surrogate
microorganisms, and they also addresses the need for
performance testing of treatment units to evaluate
inactivation efficacy of surrogate microorganisms.12 Pathogen
inactivation efficacy of emerging resource recovery
technologies have been evaluated over the past few years;
pathogen inactivation and biocidal factors have beensummarized in previous reports.14–16 However, individual
technologies are yet to be characterized for implementing the
HACCP approach in SSP. To fully implement HACCP-based
management in SSP, the following procedures are needed fortechnological barriers in advance:8,17 1) identification of
CCPs in a sanitation system, 2) selection of monitoring
parameters that affect pathogen inactivation, 3) developmentof a pathogen inactivation kinetics model, and 4)
determination of the CLs for sufficient inactivation ofpathogens.
This review aimed to identify the potential CCPs among
resource recovery technologies used in a non-sewer sanitation
system, and to identify the monitoring parameters at CCPs.
First, we evaluated the barrier efficacy of individual
technologies based on storage/operating time to reduce theconcentration of the surrogates to the target log reduction
values (LRVs). The inactivation rate of pathogens is
influenced by physicochemical factors of matrices.17
Therefore, CLs of storage time and environmental factors
were determined using regression tree which allowed tocategorize the required storage time according to theenvironmental condition of storage. This review provides
with the potential CCPs in a non-sewer sanitation system,
what to be monitored at CCPs, and the concept ofdetermining CLs which needs to be achieved at CCPs. The
concept provided in this review assists engineers andpractitioners implementing the HACCP-based microbial risk
management which facilitates recycling of nutrients inhuman excreta to agriculture.

feces,18 and regulated to <103 cfu g−1 feces;6,18 thus, 6 log10
inactivation is required. The concentration of helminth eggs
in fecal matters is 4 log10 eggs per g feces,18 and viable egg
should not be detected,6 accordingly 4 log10 inactivation isrequired. Pathogens originated from misplaced feces are
diluted by 10 000-fold in source-separated urine,6 therefore,
the target inactivation for urine is smaller than fecal matter:5 log10 unit of viruses and phages, and 2 log10 unit ofbacteria. For helminth eggs, we set a 1 log10 unit as aconservative target.

2. Methods
2.1 Target log reduction values
The target LRVs are shown in the Table 1. For viruses andphages, the target LRVs were set to 9 log10, according to thesuggestion in the 2006 WHO Guidelines.6 Bacterial
concentrations in feces were approximated to 9 log10 cfu g−1

Table 1 Target inactivation values (log10(Nt/N0)) of virus/phage, helminthegg, and bacteria in urine and fecal sludge, determined according to the2006 WHO requirements

Virus and phage
Helminth egg
Bacteria

In urine

In fecal sludge

5
1
2

9
4
6

1010 | Environ. Sci.: Water Res. Technol., 2023, 9, 1008–1029

2.2 Estimation of storage duration to achieve the target log
reduction values of microorganisms
This study estimated time to achieve the target LRVs basedon the data reported in previous studies. We extracted timecourse concentrations of microorganisms when they were
provided in a table (i.e., numerical form). Data extraction
from a figure was carried out using the WebPlotDigitizer
version 4.2.19 Time-course concentration of microorganisms
were applied to the Hom's model.20ln(Ni/N0) = −ktm,(1)where Ni and N0 are the concentration of microorganisms attime i and 0, k is the inactivation rate constant, and m is theempirical constant. Storage duration to achieve the target
LRV was derived as follows:

1
 m
1
LRV
t¼
ln 10
;
(2)
k
where k and m are the estimated parameters of Hom's model.
When the k value was provided, we used it to estimate
storage duration to achieve the target LRVs. The parameter
estimation was performed based on the maximum likelihood
approach using the statistical software R (version 3.6.1)21 (seeESI†).
2.3 Regression tree
Regression trees were created using the rpart package in thestatistical software R.21 The explanatory variables employed
in a regression for stored urine were pH, temperature,
dilution ratio, and those employed for stored fecal sludge
were pH, temperature, moisture content, uncharged
ammonia concentration, and types of desiccants. The
number of branches was determined based on the complexityof parameters to generate minimum error in cross-validation.

3. Sanitation technologies for agriresource recovery
3.1 Urine
Urine contains the largest proportion of nitrogen (90%),
phosphorus (50–65%), and potassium (50–80%) of the feces
and urine fractions released from the body.22 In fresh
urine, approximately 85% of nitrogen present in the form

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of urea, while urea is hydrolyzed to ammonium by urease
produced by urease-forming bacteria during collection andstorage.23 Simplest management is storage and direct
application of the urine, however the concentration is low,
about 0.6%, large volumes need to be transported, stored
and applied when reused. Several treatment technologies toremove the water to produce a concentrated fertilizer has
been developed during recent years. Recovered nutrientsvary depending on technologies. Nearly all nutrients arerecovered by nitrification/distillation, while phosphatemineral is the primary nutrients in struvite.24 The loss ofnitrogen is prevented by addition of acid or alkali because
it decreases urease activity and inhibits urea hydrolysis.25,26
Recovered urea (80–90% of the N in urine) is used to
produce the slow-release nitrogen fertilizer which allows
effective
utilizationofnutrients
inagricultural
production.26

Feces potentially contain a large number of pathogens.
The fecal contamination of source separated urine wasestimated to be within a range of 1.6–18.5 mg of feces per
little of urine, with a mean of 9.1 ± 5.6 mg L−1, thus resulting
in about a 5 log lower concentration of pathogens than infeces.6,27 The main risk is from organisms like Mycobacterium
tuberculosis that can be excreted in the urine.28
3.1.1 Storage. Urine is normally not used immediately but
stored in containers or tanks. Heterotrophic bacteria grow infresh urine consuming the nutrients and organic matter.29–31
During storage in a sealed container, urea in urine isenzymatically degraded to ammonia and carbon dioxide,
resulting in increased pH to more than 8.8.32 Ammonium
and carbonates, which inherently present in excreta matrices,
promote inactivation of single-stranded RNA phage by
degradation of viral genomic RNA via alkalinetransesterification.33

Fig. 2 (a): Storage time of urine to achieve the target log reduction values for Gram-negative bacteria (G−), Gram-positive bacteria (G+), Ascaris
eggs (helminth), phages, and viruses.29,31,33,34,36,38,39 (b): Regression tree to determine storage time of urine to achieve the target log reduction
values for phages and Ascaris eggs.29,31,33,34,36,38,39

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Double-stranded DNA phages, including 28B31,34 and T4,35
rhesus rotavirus,34 and Ascaris eggs,36 are the most tolerant
surrogates in urine. The estimated storage time to achieve
the target LRV is the longest for Ascaris eggs and phages
under the physicochemical conditions reported in literatures(Fig. 2(a) and S1†); thus, they were found to be the criticalsurrogates within those environmental conditions. Pathogen
inactivation in stored urine is mainly governed by ammonia
and carbonate concentrations, pH, and temperature.31,33,37,38
The storage time for 1 LRV of Ascaris eggs and 5 LRV ofphages were determined primarily based on storage
temperature; it was approximately 25 d when the temperature
was >24.5 °C irrespective of the dilution ratio (Fig. 2(b)). At
temperatures >14.5 °C and without dilution, 75 d of storage
was sufficient for inactivation, while six months of storage
was needed for persistent phages (Fig. 2(b), node 3). This
indicates that the WHO recommendation of 6 month storageat >20 °C ensures sufficient inactivation in most cases.
However, when urine was diluted with water, the inactivation
rate was slower, and the storage time was exceptionally long
(Fig. 2(b), node 10), probably because of the lower
concentration of biocidal components, including bases,
indigenous microbes, and enzymes.35,39 Rotavirus and phage
28B were not inactivated in six months at 5 °C,34 therefore
they are not included in the Fig. 2. The storage period should
be monitored in a cold region or during winter if external
heating equipment is not available. It may not practical tostorage urine for such long times at a scale in dense urban
environments due to storage volume requirements.
Additional treatments are needed to shorten storage time.
The strategy for urine storage should also consider theamount of water used for flushing, as it affects theconcentration of biocidal components in urine. Additionally,
if diluting flush water is decreased during collection the
agricultural value increases as less water need to be managed
and applied to the fields.
3.1.2 Solar heating and pasteurization. An increase intemperature enhances pathogen inactivation; therefore,
thermophilic storage would help shorten the storage time.
Solar heating of urine containers is a relatively simple
measure to increase storage temperature. Moreover, solar
heating gives the added advantage of ultraviolet radiation
when a transparent container is used; the container
temperature can be increased to 65 °C in a tropical climate,which promotes pathogen inactivation.40 At thermophilictemperature, bacterial inactivation occurs via damages on
metabolic functions, cell membranes, and enzyme activities;
while viral inactivation occurs due to the loss in ability tobind with its host cell, which probably caused by structuralchange in proteins.41,42 Increasing temperature can indirectly
facilitate helminth eggs inactivation by increasing themembrane permeability of the lipid layer on the egg shell.43
Although thermophilic conditions are not achieved incolder regions, pathogens are inactivated faster undercontinuous sunshine, and the required storage time for
Ascaris inactivation under sunlight is halved compared tothat for storage indoors (light intensity was not reported).44
Sunlight intensity changes with the latitude, which affect theinactivation efficiency of pathogens in stored urine;45
therefore, regional difference in the sunlight intensity need
to be considered. Irrespective of the presence of sunlight, theestimated times for the target LRVs were 17 d and one d for
Ascaris eggs and Gram-negative bacteria, respectively, while
those for bacteriophages MS2, phiX174, and 28B were 41,
185, and 275 d, respectively (Table S1†). The persistence ofviruses, phages, and helminth eggs during thermophilicstorage has not been reported in the literature. Phages,
specifically 28B, phiX174, and T4, could be conservative
indicators of urine pasteurization because the storage timerequired to reach the target LRV is longer than that forhelminth eggs. The estimated storage time was longer than
two months at temperatures >34 °C without sunlight
exposure (outlier in the Fig. 2(b), node 3).
3.1.3 Nitrification and distillation. All nutrients inhydrolyzed urine are concentrated in a small volume via theprocess that consists of nitrification and distillation.
Nitrification is applied to reduce nitrogen loss throughammonia volatilization, and the stabilized urine is thenconcentrated by distillation, which enables complete nutrient
recovery from source-separated urine.24 The complete
nutrient recovery process requires relatively high electricalenergy input and investment costs: a specific energy
consumption of the distiller is 100 W h L−1 treated urine,
while the energy demand for nitrification is about 50 W h L−1
urine.46
Pathogens are inactivated during a 20 day-nitrification at
20 °C and pH 6.05: bacteriophage Qβ is inactivated by 2 log10by physical (i.e., aeration) process, and Salmonella
typhimurium and Enterococcus spp. are inactivated by 1 log10
by biological (i.e., microbial activity) processes.47 However,
nitrification is not a stand-alone process for sanitization
because of the insufficient removal of pathogens; thus, itcannot be a CCP for monitoring. Pathogens are effectively
inactivated during distillation as they are subjected tothermophilic conditions (∼80 °C).24
3.1.4 Membrane technologies. Membrane technologies
such as membrane distillation, nanofiltration, reverse
osmosis, and forward osmosis are applied to concentratesource-separated urine on-site. Pathogens are retained in thefeed solution as they cannot pass through the membranefilters; the fate of the pathogens could then be determined in
the concentrated urine. Forward osmosis technology isadvantageous in regards to operational cost and equipment
investment in comparison with nanofiltration and reverse
osmosis due to the lower hydraulic pressure and energy
demand.48
Previous studies have simulated the fate of E. coli andMS2 in a feed solution using concentrated synthetic
urine.30,49 MS2 was inactivated more slowly than E. coli, andthe time for a 5 log10 inactivation was estimated as 4.8
months and 5 h for 10-folds concentrated non-hydrolyzed
and hydrolyzed urine, respectively. Ammonia activity of 10-1012 | Environ. Sci.: Water Res. Technol., 2023, 9, 1008–1029

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fold concentrated urine was approximately 1000 mM.
Ammonia activity in urine facilitated the inactivation of MS2
better than other parameters such as osmotic pressure andpH,25 suggesting that concentrated urine should becompletely sealed to avoid the loss of ammonia duringstorage. The fate of persistent pathogens, including helminth
eggs and DNA phages/viruses, should be investigated infuture studies.
3.1.5 Struvite precipitation. Struvite (MgNH4PO4·6H2O) isa fertilizer obtained in powder-form from source-separated
urine (capturing almost all the P and about 5% of the N).
Struvite crystals form when the supersaturation in thesolution is achieved by magnesium addition, and then they
are recovered in a powder form through filtration and drying.
The fate of the pathogens during struvite production has
been reported in previous studies.50,51 Phage phiX174 andAscaris eggs were not considerably inactivated during themixing of influent urine (pH 8.8) and during filtration, while
Ascaris eggs and bacteria accumulated on the cake layer
during precipitation. In the subsequent 3 day drying at 5–35
°C, the Ascaris eggs and phage phiX174 in the cake were notsignificantly inactivated.52 A 2 log10 inactivation of Ascaris
eggs was achieved in less than one day, whereas three days
were needed for a 3.5 log10 inactivation of phiX174 at 36 °C
and 35% relative humidity, which indicates that viruses
remain infectious for a longer duration in struvite cakes.52
Initial heating prior to drying or drying at higher
temperatures could prove effective in inactivating pathogens;however, the temperature should be maintained at 55 °C orless to prevent substantial loss of ammonia. Struvite
production can be a CCP if pre-heating of stored urine or
post-drying at thermophilic temperatures is employed.
3.1.6 Alkali dehydration. Alkali dehydration is used toreduce the volume of source-separated urine through drying
in alkaline matrices, such as lime and wood ash. Liquid
urine is converted into a dry, solid fertilizer. Urease activity is
inhibited under alkaline conditions (pH > 10), which
prevents the loss of nitrogen due to volatilization ofammonia generated through urea hydrolysis.53
We estimated the required retention time for sanitization
using the data extracted from Senecal et al.54 Bacteria andphage were inactivated post-storage in less than three days at
20 °C, whereas the inactivation of Ascaris took longer than
five months. The storage time for Ascaris inactivation can beshortened to four days by heating at >42 °C. Thermophilicstorage of dried urine is recommended when the prevalenceof helminth infection is high.
3.1.7 Ion exchange/sorption. Sorbents, including charcoal
and mineral sorbents, are used for the absorption and ion
exchange of nutrients in urine.5 Charcoal, especially activatedcarbon, has the potential to remove viruses, bacteria, andbacterial spores during water treatment, although theremoval efficiency is not always high.55–57 To the best of ourknowledge, no studies have been conducted on the fate ofpathogens in the sorption and ion exchange processes.
Ammonium, phosphate, and potassium ions are recovered by

the use of ion exchange resins. Those adsorbed ions aredesorbed in solutions containing NaOH, H2SO4, or HCl.
Pathogens may be physically adsorbed on the sorbents,
however, removal efficiency of viruses by adsorptions is lower
than 5 LRVs.58–60 Pathogens can be inactivated in the NaOH
desorption solution, while they may not be inactivated inacid desorption solutions. The fate of pathogens in generated
solutions and sorbents need to be investigated.

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3.2 Fecal sludge
Urine contains most nitrogen and about half of phosphorus
and potassium in human excreta, while feces are rich inphosphorus and potassium, and contain most carbon
excreted from the body. Fecal sludge is the fecal waste stored
within onsite sanitation systems. In addition to fresh feces, it
includes everything that go into toilet pit, including urine,
anal cleaning materials, greywater, and municipal solid
waste.61 Although the physicochemical characteristics aredifferent from stored feces, fecal sludge also contain organic
matter and nutrients; therefore, it is used as combined soil
amendments and fertilizers. The concentrations of nitrogenand phosphorus are 10–100 times and 2–50 times higher
than those of domestic wastewater.61 The available nutrientsare affected by the type of toilet pit and treatment
technologies, for example, treatment takes place in an open
system is prone to lose nitrogen through volatilization andleaching.
Untreated fecal sludge has a high oxygen demand due tothe presence of readily degradable organic matter.
Stabilization process is recommended to avoid depletion ofoxygen in farmlands. In a centralized system, fecal sludge iscollected from a single pit, transported to a (semi-)centralized
treatment facility, and treated before its application in
farmlands.62
3.2.1 Storage. Some containment technologies, including
bucket latrines, pit latrines, and septic tanks, are notdesigned for resource recovery, but fecal sludge collectedfrom them can be applied for resource recovery if it iscollected and transported accordingly.63 Pathogens areaffected by biocidal components inherent in human excreta
matrices along with external environmental factors and are
therefore inactivated during storage, which is often called
self-sanitization or natural inactivation.
The estimated storage time was longest for phages andAscaris eggs under the physicochemical conditions reported
in the literature (Fig. 3(a) and S2†). Ascaris eggs can remain
viable for more than one year at temperatures below 22.5 °C(Fig. 3(b)), whereas in a warmer climate with lower moisture
content (<26%) and with the use of desiccants, they can beinactivated within 1.5 months or less (Table S2†).
Chemical or physical treatments such as ash/lime,
pasteurization, and solar drying are commonly used toshorten storage time. A pH > 9 has been recommended by
WHO,7 but the target LRV for MS2 and phiX174 may not be
achieved in six months at pH > 9 (Table S2†). Recent studies

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Critical review

Fig. 3 (a): Storage time of fecal sludge to achieve the target log
reduction values for Gram-negative bacteria (G−), Gram-positivebacteria (G+), Ascaris eggs (helminth), phages, and viruses.81–83,144,145(b): Regression tree to determine storage time of fecal sludge toachieve 4 log reduction of Ascaris eggs.81–83,144

have suggested that the matrices pH should be maintained
above ten along with the presence of ammonia for virus andbacterial inactivation.14,64 Complete sealing of the containeris effective in reducing the volatilization of ammonia and theinactivation of pathogens. The pH and ammonia
concentration need to be high enough for hampering thebiological activity otherwise it may lead to accumulation ofharmful gasses such as methane and sulfur dioxide; thisfurther leads to oxygen deficiency. Sanitation workers are at ahigh risk of exposure to harmful gases and anoxic conditions
when they empty a pit.65 Inhalation of harmful gases underanoxia must be prevented by providing active ventilation or
should be monitored using an oximeter. Trade-offs inchemical and microbial risk should be addressed.
Although storage is a potential CCP, the sanitization
efficacy is mainly affected by storage time, temperature, and1014 | Environ. Sci.: Water Res. Technol., 2023, 9, 1008–1029

Environmental Science: Water Research & Technology
the use of additives, which is different by the type ofcontainers. The risk of exposure to pathogens is determinedby the frequency of collection/emptying of containers, andthe required LRVs can be changed by the type of containers
and the collection schemes of containers, consequently. The
biocidal factors in fecal sludge are common in container
storage, while controllable biocidal factors vary depending on
the structure and the type of containers. Thus, we havereviewed pathogen inactivation during storage in different
types of containers in the following sections.
(1) Bucket latrine and container. A bucket latrine consists ofa pedestal or seat drop hole with a bucket or pan (25–30 L)
placed in a chamber below. When the bucket is full, it ismanually removed and emptied66 (Fig. S3†). The installationof bucket latrines should not be promoted because of thesignificant infection risk through vector breeding, humancontact while emptying and washing a bucket, and overflow
due to inadequate regular emptying.66 Container-based
sanitation (CBS) involves the use of a sealable container
placed under a pedestal and a collection and transportation
system for containers; it is a promising option for resource
recovery in a densely populated area.67 When urine-diverting
toilets are used for households in a densely populated area, aCBS system realizes off-site resource recovery from thecollected urine and feces.
The pathogen inactivation rate in a container depends on
the storage time without the application of chemical/physicaldisinfection. Significant inactivation can occur when stored
buckets are heated from being directly exposed to sunlight.66
Chemical disinfectants are used where bucket-based
sanitization of human excreta matrices is needed, specificallyin emergency situations such as infectious disease outbreaksin evacuation shelters. Lime-based sanitization with calcium
hydroxide resulted in approximately 5 log10 inactivation of
fecal coliforms, intestinal enterococci, somatic coliphage, F+specific phages, and Bacteroides fragilis phages in 30 min at a
pH > 12,68 while nematodes are not inactivated during thisshort treatment.69 Because of the limited storage capacity
and consequent shorter storage time compare to pit latrines,
significant inactivation of pathogens is not achieved.
Treatment in buckets and container can be a CCP if
additional onsite sanitization is combined.
(2) Single pit latrine. A single pit latrine is a shaft dug
below the ground level, which is either lined with reinforcing
materials or left unlined.66 A single ventilated improved pit(VIP) latrine is associated with a ventilation pipe that vents
odor and acts as a trap for flies (Fig. S4†). The potential forfly breeding is reduced by a fly mesh at the ventilation pipe,
the use of a toilet cover, and frequent addition of bulking
materials or ash to reduce the possibility of flies
encountering fresh fecal material. Excreta, along with anal
cleaning materials (water or solids), were deposited in a pit.
Depending on how deep they are dug, some pits may last for
more than 20 years without emptying, which allows greater
inactivation of pathogens during longer storage than bucket
latrines and CBS systems.70 However, in a dense area, pit

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emptying is periodically needed owing to the high rate of pit
filling and space limitations. Improper emptying is
associated with a significant risk of infection among
sanitation workers.71
Pathogen survival in a pit latrine is affected bytemperature, moisture content, oxygen availability, andpresence of indigenous microorganisms.72 Uncharged
ammonia has a biocidal effect; however, the sludge in a pit
latrine has neutral pH, which is more conduction for ionizedammonia than uncharged ammonia.73 Additionally thedesign with good ventilation results in losses of the formed
ammonia to ambient air. The temperature in a pit latrinechanges when there is a change in ambient temperature.74
Because the temperature within a pit latrine is not commonly
controlled, the best monitoring parameter is the storagetime.
(3) Septic tank. A septic tank is a watertight chamber madeof concrete, fiberglass, polyvinyl chloride, or plastic throughwhich blackwater and greywater flow for primary treatment
in one to three chambers (Fig. S5†).62 The treatment
performance is determined by maintaining an appropriate
hydraulic residence time for the solid–liquid separation ofwastewater. The effluent from the final chamber is further
treated using a subsurface drain field or other additional
processes.
The mechanisms for the reduction of pathogens in a
septic tank include settling with sludge and natural
inactivation. The efficacy of pathogen reduction in a septictank varies depending on the type of pathogen and isaffected by the temperature and retention time.75 Predationby heterotrophic microorganisms, interspecific competition
for nutrients and reduced red-ox potential are also involvedin pathogen inactivation.76,77 The inactivation efficiency ofpathogens in a septic tank is usually low; therefore, highlevels of pathogens remain in both the effluent and sludge.
Helminth eggs are removed from the liquid phase by gravity
settling because they are sufficiently large for settling to beeffective, but the reduction is usually less than 0.5 log10.6(4) Twin pits for pour flush toilet (wet pit). The twin pits forpour flush toilet consists of two pits connected to a pourflush toilet (Fig. S6†). Unlike the double VIP and FossaAlterna which are described in the latter section, this toilet
allows the use of water and does not require addition oforganic materials. One pit is used while the fecal contents inanother pit is stabilized.66 Because fecal sludge has
undergone significant dewatering and degradation, further
treatment in a semi-centralized treatment facility is not
required. After one year of storage in a pit, significant
inactivation of viruses, bacteria, and protozoa is achieved,
but Ascaris eggs may still be viable.78 Fecal sludge istransformed into nutrient-rich humus that is safe to excavate
after storage for more than two years.66 There is a higher riskof groundwater contamination due to more leachatecompared to waterless toilets.
(5) Double alternating dry pit (dry pit). Consisting of twopits, a double alternating dry pit allows at least 1.5 to 2 years

resting time of the pit contents while another pit is used (Fig.
S7†). The excreta in the pit is transformed into nutrient-richhumus. Fossa-Alterna, an alternating dry pit, requires the
addition of leaves and soils following each defecation to
introduce microorganisms that assist in the stabilization of
the excreta. Owing to enhanced biological degradation,
sludge in the pit is transformed into an earth-like material
that is used as a nutrient-rich soil conditioner (mainly P as
most of the N is lost during storage) after one year ofstorage.62
Pathogen inactivation and other important factors are
similar to double dehydration vaults, which are described in“(6) Double dehydration vaults (double desiccating toilet)” in
the present section. The distinctive feature of the double
alternating dry pit is that urine is not stored separately,
which could result in a high concentration of ammonia in
the pit. The presence of indigenous microorganisms also
affects pathogen inactivation in pits. Vector attraction andodor are significantly reduced by installing ventilation pipes
compared with non-ventilated toilets. The constant additionof ash, lime, soil, or sawdust further reduces the moisture
content, minimizes odors, and provides a physical barrier
between the excreta and vectors. The addition of alkalimaterials, such as ash and lime, promotes pathogen
inactivation and dehydration.
Viruses, bacteria, and helminth eggs survive in pits forseveral months under mesophilic conditions.79 WHO
recommends the common practice of double alternating dry
pits and double dehydration vaults in the SSP. A storage timeof 1.5–2 years at ambient temperature (2–20 °C) is
recommended in areas with a high prevalence of helminth
infections.6 The recommended storage time can be shortened
to one year in tropical climates with ambient temperatureshigher than 20–35 °C.6 A prolonged storage time of 18
months is recommended for highland subtropical areas (17–20 °C).66
When subsequent solar drying is used, the storage time
can further be reduced to 8–10 months.66 When the pit
content was maintained at pH > 9, the storage time wasshortened to six months.6 Recent studies have suggested thatthe pH of the matrices should be maintained above ten in
the presence of ammonia for effective inactivation of virus
and bacteria.14,64(6) Double dehydration vaults (double desiccating toilet). A
double dehydration vault, which is associated with a urinediverting dry toilet, generates dehydrated feces that can beused as a soil conditioner (Fig. S8†). Commonly usedadditives include ash, dry soil or sand, dry leaves, dry grass,rice husks, lime, crushed oyster shells, and shea nut shells,
all of which contribute to the essential attributes ofgenerating dehydrated fecal sludge that is bulky and dry.80
Pathogen inactivation through urea amendment has been
evaluated in source-separated feces.36,81–83 Inactivation of
Ascaris eggs is faster in feces amended with a mixture of ash
and urea than that with a single addition of ash or urea.36
Cruz et al.81 demonstrated that greater inactivation of Ascaris

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eggs was achieved with a lower moisture content (27.5%).
When 1–2% dirt and urea were added to feces, viable Ascaris
eggs were reduced by 2 log10 in 14 d at 28 °C, whereas theeggs were viable when feces was stored without the additionof urea. A higher storage temperature further promoted theinactivation of Ascaris eggs,36,81 Enterococcus spp., Salmonella
spp., and phage 28B, while the inactivation rate is not linearwith temperature.83
3.2.2 Alkali sanitization. Ash and lime (limestone, slaked
lime, and burnt lime) are common additives that have been
applied to a variety of sludges, including sewage sludge,
dehydrated feces, or humus, and finished compost. Alkali
additives are used to minimize the potential for odor
generation, inactivate pathogens, and reduce the material's
vector attraction potential.84 Common desiccants applied todehydration vaults and desiccation toilets including woodash (pH: 9.4 to 11.3), rice husks (pH 10.6), and lime (pH: 10.3
to 12.4) have a basic pH.80 The application of alkali treatedfecal sludge is effective to neutralize acid soil. The increaseof pH of the soil amended with slaked lime alters microbialcommunity structure and the enzymatic activity, whichfacilitates nitrogen mineralization in the soil.85,86 The
nitrates-uptake capacity of a plant should be considered toprevent surface and groundwater contamination.
Alkaline-treatment of compost facilitates inactivation of E.
coli via the damage to the outer membrane and enzyme
activities, while it leads to the loss of infectivity of coliphage
via the partial capsid damage and RNA exteriorization.87 In
addition to the single effect of increasing the pH, alkali
additives change the ratio of indigenous biocidal
components, including ammonia and carbonate, which
enhances the biocidal effect of ammonia inherent in excreta
matrices.
The effective lime dosage varies from 1% to 35%, and it
elevates the pH from 8.0 to 12.9.68,69,87–89 The estimatedstorage time for inactivation of phage MS2 was several hours(Table S3†), while it took more than six months forinactivation without desiccants (Table S2†); this indicatedthat lime treatment is a promising measure for virus
inactivation. The estimated time for inactivation of Ascaris
eggs was three weeks in the presence of approximately 500
mM NH3, but it took more than two years with 50 mM NH3,
which suggests that alkaline pH alone has a limited effect onhelminth inactivation, as shown by Senecal et al. with no
reduction at all in ammonia free pH 12.4 environment.90 The
tailing-off profiles resulted in exceptionally longer estimates(more than two years for Ascaris, two months for E. coli, and26 d for MS2) (Table S3†).
It should be noted that the estimates do not include
datasets obtained at lower temperatures (<20 °C) (Table S3†),
although surrogates are found to survive for several years at
lower temperatures.91 Future studies should evaluate theeffects of alkali additives in cold regions. Lime treatment
should be designed to inactivate Ascaris spp., which survives
the longest under alkaline environments; when Ascaris is not
an organism of concern, a pH > 10 is sufficient for theinactivation of viruses and bacteria.64 Viruses and phages are
rapidly inactivated at 4−28 °C. However, the virucidal effectof lime is yet to be fully understood owing to the lack of datafor phages and viruses.64 Future studies on lime treatment
should characterize the inactivation of ssRNA and non-ssRNA
phages and assess whether phages are representative of theinactivation of pathogenic mammalian viruses. By using
burned lime, CaO, the oxidation reaction is very strong andreleases heat together with increased pH. During emergency
sanitation, this can be an alternative. However, the highreactivity poses a health risk to the workers mixing in the
lime.
3.2.3
Ammonia/urea
sanitization.
Ammonia/urea
sanitization, such as Peepoo bags, is applied at theindividual, household, and city levels.92 When the use ofsynthetic urea and ammonia solutions as additives in urinediverting toilets is not economically feasible, stored humanurine may be a potential alternative. An ammonia solution
and urea pellet take advantage of the long-lasting sanitization
effect in high organic-loaded matrices and the addition offertilizer value. Urea is hydrolyzed to ammonia and carbonate
by urease-producing bacteria, which exist ubiquitously in thesurrounding environment. The pH of the matrices increases
with the production of ammonia, and at pH greater than
9.25, which is the pKa of ammonia (25 °C), the fraction ofuncharged ammonium becomes dominant in theequilibrium of ionized ammonium.
Kohn et al.64 reviewed the inactivation profiles ofsurrogates and proposed treatment recommendations forammonia sanitization. The recommended storage times are
six months (T < 20 °C), two months (T < 30 °C), and 15 days
(T > 30 °C) with 1.5% urea addition (corresponding to 0.75%
NH3) at pH > 8.8.64
Treated fecal sludge is used as a quick-release nitrogen
fertilizer which has nitrogen as a form being immediately
available to plant roots. Oversupply of quick-release fertilizers
to farmland leads to nitrates pollution of surface andgroundwater due to the lower nitrates uptake efficiency byplants, and consequent washing out of the root zone ofplants.93 Direct application of treated fecal sludge should bedesigned to prevent contamination of drinking water sourcewith nitrates.
3.2.4 Solar drying and pasteurization. Solar drying is
widely applicable for the stabilization of solid waste
including feces, excreta, compost, and fecal sludge; it can beused for an onsite or a centralized treatment (e.g., dryingbed). An unplanted or planted drying bed is used for volume
reduction of fecal sludge collected from a community-level
facility, such as pit latrines and septic tanks.62 Pathogen
inactivation in a sludge drying bed has been summarized ina previous study.66 There was a reduction in the total numberof pathogens during the drying process, but theconcentration of pathogens increased with a reduction in thevolume of the sludge. Although solar drying on a drying bed
is effective in reducing sludge volume, sufficient degradationof organic matter and pathogen inactivation were not1016 | Environ. Sci.: Water Res. Technol., 2023, 9, 1008–1029

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achieved during the 15 day drying period.62 Further
treatments such as storage and composting wererecommended since the increased dry matter content resultin longer survival of many microorganisms mainly due todecreased heat transfer capacity in dried substrate.94
Irrespective of the presence of sunlight, the target LRVs
for bacteria and helminth eggs were achieved within fourdays at a thermophilic temperature (50–75 °C), but it should
be prolonged at a mesophilic temperature; the estimate for
Ascaris inactivation is eight months at 31 °C (Table S4†).
Helminth eggs are appropriate indicator microorganisms forsolar drying. Future studies should include inactivation ofphages and viruses at mesophilic temperatures with exposure
to sunlight.
3.2.5 Composting. Composting is primarily an aerobic(and sometimes anaerobic) microbiological decomposition
process that stabilizes organic matter to odorless
substances.80 Increasing the temperature to >40 °C in thethermophilic stage effectively inactivated pathogens. Even if athermophilic temperature is not achieved, a small change intemperature in the mesophilic range facilitates theinactivation of Ascaris eggs.95 Pathogen inactivation in acomposting chamber primarily relies on the heat generated
by aerobic biological reactions. Thus, maintaining optimum
conditions for biological reactions is important for
sanitization. Alkaline additives are used to enhance pathogen
inactivation; however, an excessively high pH in acomposting toilet may limit the overall biological reactions
occurring within the pile of excreta to sufficiently raise thetemperature.80
Under optimum conditions for composting with organic
waste, the temperature of the chamber reachedapproximately 65 °C over several days, and the thermophilic
condition was maintained for several weeks. The estimatedcomposting duration for sufficient inactivation of E. coli andEnterococcus spp. is less than two weeks, as long as thethermophilic temperature of 50–60 °C is maintained.96
Bacterial regrowth can occur when the chamber temperatureis <50 °C.96 The fate of coliphages (T4, λ, Qβ and MS2) insawdust was investigated under various matrix temperaturesand moisture content.97 The estimated time for T4
inactivation was 126 d at 30 °C and 15 d at 50 °C, while thatfor Qβ inactivation was 15 d at 30 °C and three days at 50 °C.
Helminth eggs and Salmonella can survive longer thanphages under thermophilic conditions. Ascaris eggs werecompletely inactivated under an 8 week co-composting as
long as the thermophilic temperature (50.7–58.7 °C) was
maintained for 31 d, and pile turning was conducted every
seven days.98 Based on the modeling study by Vinnerås et al.,Salmonella is considerably more resistant to temperaturecompared to Ascaris.99
A composting latrine chamber was originally designed fora 6 month storage period to achieve adequate pathogen
removal. However, it is now recommended to increase thestorage time to 1.5–2 years,100 because composting chambersin the field were unable to reach a high enough temperature

for the inactivation of resistant pathogens such as
helminths.101
3.2.6 Vermicomposting. Vermicomposting is a mesophilic(<35 °C) waste oxidation and stabilization process in which
earthworms and diverse microbial communities jointly
process organic waste, including source-separated humanfeces, fecal sludge, and human fecal slurry under aerobic
conditions.102,103 Vermicomposting is available on anindustrial scale for centralized treatment and on a toilet
chamber scale for decentralized treatment. The earthworms
function in synergy with bacterial communities within a“vermi-filter”, which treats diluted domestic wastewatersludge in a system inoculated with earthworms.104
Pathogen inactivation is achieved by the intestinal
digestive action of earthworms as well as intestinal enzymes
and antibacterial coelomic fluids excreted by earthworms.103
Pathogen inactivation is indirectly promoted by enhanced
aeration by the burrowing activity of earthworms, whichfavors other microbial communities that compete foravailable nutrients by engaging in antagonism andphagocytic activity, as well as in the production of humic
acids.103 Vermicomposting cannot be performed atthermophilic temperatures of composting. Temperature is an
important factor for worm activities.
Previous studies have investigated the reduction inpathogens during vermicomposting. More than 2 log10
removal of coliforms and 1.6 log10 removal of E. coli wereachieved after 60 d of vermicomposting.105,106 Viable Ascaris
eggs were not reduced after 90 d of vermicomposting,whereas helminths, coliforms, and Salmonella were
inactivated after 180 d.107 Inactivation of thermotolerant
coliforms, Salmonella spp., Enterococcus spp., andbacteriophage phiX174 is limited to 1.5 log10 to 2 log10.108
However, further treatment (e.g., storage for longer than oneyear) is necessary to meet the requirements of the 2006 WHO
guidelines.14,108 Vermicomposting is not a stand-alone
process for sanitization because of the insufficient removal ofpathogens; thus, it cannot be a CCP for monitoring.
Pathogen inactivation is affected by stocking density ofworms and the pH of the matrices. Optimum stocking
density of earthworms (2.0–4.0 kg m−2) should be maintainedfor pathogen inactivation.103 A greater stocking density
beyond a certain level led to overcrowding of earthworms,
which resulted in a decline in waste processing efficiency
and consequently affected pathogen inactivation. In
vermicomposting source-separated human feces, inactivationof Enterococcus spp. and thermotolerant coliforms is affectedby pH. The compost pH is also correlated with the maturity
index; thus, the maturity of vermicompost is involved inbacterial
inactivation.108
Meanwhile,forcoliphage
inactivation, the time since last maintenance appears to be
more important than pH and ammonia concentration.108 The
pH of vermicompost and composting duration should be themonitoring parameters in vermicomposting.
3.2.7 Black soldier fly composting. Black soldier fly (BSF)(Hermetia illucens) larvae grow by consuming organic waste,This journal is © The Royal Society of Chemistry 2023

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including animal manure and fecal sludge, which results in
up to a 75% volume reduction of organic waste, together withnutrient removal such as nitrogen and phosphorus.109 The
residual fecal sludge after the BSF larval feed must becomposted or anaerobically digested to produce a soil
conditioner.110
Adenovirus, reovirus, and enterovirus (106 TCID50 g−1)
were inactivated to below the detection limit in two weeks,
and more than 6 log10 inactivation of Salmonella spp. wasachieved in seven weeks, whereas, Enterococcus spp. andAscaris suum eggs were not reduced at eight weeks.111
Pathogen inactivation in BSF composting is mainly timedependent, through natural inactivation at an ambient
temperature, although adsorption onto particles andammonia inactivation occur. Retention time should be an
appropriate monitoring parameter in a BSF composting
system. BSF composting is not a stand-alone process for
sanitization; thus, it cannot be a CCP. Additional sanitizationis needed, especially where helminth infection is a mainconcern.
3.2.8 Lactic acid fermentation. Lactic acid fermentation(LAF) is a biological anaerobic process. The advantage of LAF
in human excreta is its ability to prevent nitrogen loss andinhibit the production of malodorous compounds, which is
attributable to suppressed protein degradation during
fermentation. LAF is a fundamental process in the terra preta
sanitation system, in which human excreta are converted into
terra preta-like soils and subsequently used in agriculture.112
LAF does not induce a temperature increase; fermentation
temperature is usually below 36 °C. Pathogen inactivation
through LAF is attributed to the acidification caused by the
production of lactic acid as well as the release of othercompounds with suppressive effects on pathogens such asbacteriocins, glucose oxidase, and hydrogen peroxide.113
Inactivation of E. coli in a week varied from 0.1 log10 to 4.4
log10 in previous studies;114,115 this is probably due tofluctuations in the activity of lactic acid bacteria (LAB), whichis affected by the type of carbon source (i.e., molasses orbiochar). The pathogen inactivation rate fluctuates amongexperiments conducted in a narrow range of acidic pH (pH
3.7–3.9) and even within a single study.116–118 Till date,appropriate monitoring parameters for LAF have not been
identified. The microbial activity of LAB is relevant to theperformance of pathogen inactivation; therefore, parametersthat reflect the activity of LAB can be used as monitoring
parameters.
3.2.9 Anaerobic digestion. Digestate or slurry generated
through anaerobic digestion is used as fertilizer or soil
conditioner. The determinant factors for pathogen
inactivation are the operating temperature (i.e., thermophilic
digestion at 50–60 °C or mesophilic digestion at 30–38 °C),
hydraulic retention time, pH, and volatile fatty acids.66
Without a thermophilic process, the hydraulic retention timeshould be monitored. Fully mixed systems give shortminimal retention time; therefore, these systems require
additional sanitation steps even at thermophilic temperature.

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Fig. 4 Estimated retention time to achieve the target inactivation
units of Gram-negative bacteria (G−), Gram-positive bacteria (G+),
Ascaris eggs (helminth), phages, and viruses in anaerobic digestion
under various temperatures.95,119,146–150

At a moderate temperature (28 °C), more than one yearof storage is needed for 1 log10 inactivation ofCampylobacter jejuni, whereas E. coli and Salmonella areinactivated at 77 and 35 d, respectively.119,120 At mesophilictemperatures (35−37 °C), the estimated storage time was
the longest for DNA phages (phiX174 and Bacteroides
fragilis phage B40-8), whereas the estimates for viruses(murine norovirus 1 and human adenovirus) were less than
three months (Fig. 4 and Table S5†). Ascaris eggs are notinactivated at temperatures below 36 °C without uncharged
ammonium.95,121 At a thermophilic temperature, theestimated storage time was significantly shortened to <1 dfor bacteria and Ascaris eggs and 30 d for murine norovirus
1 (Fig. 4 and Table S5†). Plug flow or batch reactors
reaching a considerably lower red-ox potential in thematerial have better reduction even at ambient treatment
temperature compared to fully mixed systems.77 When alonger retention time is not feasible due to the size
constraint of the reactor, anaerobic digestion cannot be a
CCP. A promising post-treatment is pasteurization. In
Sweden, heating at >70 °C for 1 h is required for digestate
produced from biogas plants.122 Conservative treatment
conditions should be designed to inactivate doublestranded DNA phages.
3.2.10 Drying. The latrine dehydration and pasteurization(LaDePa) pelletizer is a sludge-drying and pasteurization
technology capable of producing a dry, pelletized soil
amender in a pit latrine at a rate of approximately 1000 kg
per hour.62 The entire process of sludge drying andpasteurization was completed in 16 min. When heated above
100 °C, pathogens are completely inactivated during
pasteurization. After processing at >200 °C, Ascaris eggs (135eggs per g-TS) were completely inactivated.123
3.2.11 Microwaving. Microwave-based technology has beendeveloped for slums and emergence situations where latrines
are rapidly filled up, creating the need for efficient treatmentof fecal sludge. The sanitization performance was evaluatedover a relatively short operating times; for 100 g of fecal

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sludge, 3 min at 465 W (23 W h) compared to 1 min at 1550
W (26 W h).124,125
Pathogen inactivation occurs owing to the nonthermal(electromagnetic radiation) and thermal effects ofelectromagnetic energy.126 Microwave-based technologies cansignificantly inactivate bacteria and helminth eggs within a
few minutes.124,126 The mechanisms of bacterial inactivation
has been well summarized by Afolabi and Sohail.127 Both thethermal and athermal effects triggered by ionic conduction/
migration are involved in sanitization of fecal sludge.
Inactivation of bacteria is initiated from the rupture of cell
membranes which has a strong affinity for microwave
absorption, and subsequently leads to rupturing of the cell
walls and release of intracellular materials. The bacterial
inactivation level is determined by the input microwave
power level, amount of fecal sludge treated, and operatingtime.124 Operating conditions should be designed to increase
the amount of sludge input. A holding time of at least 3 minis recommended at the lethal temperature of thepathogens.124

molasses) contributes to maintaining a thermophilic
condition (>55 °C) without any turning or mixing for 9 d,which leads to the elimination of most of the pathogens.118
When a thermophilic condition is not achieved during thecomposting reaction, a 3 months storage of the finishedcompost79 or secondary composting at >50 °C for at least
one week7 is recommended. The addition of lime and urea,
or the application of direct sunlight can be used as posttreatment. Solar drying and heat treatment are promisingmeasures for the inactivation of helminth eggs: a 4 log10
inactivation of Ascaris eggs is achieved within a day by raising
the matrix temperature to 50 °C (ref. 128) and 75 °C.129 Eachprocess is monitored as CCPs if CLs at each process are
determined so that target LRVs is achieved in a wholeprocess train.

4. Discussion and outlook
4.1 Potential critical control points
This study reviewed the sanitization potential of resource
recovery technologies by comparing the storage time required
to achieve certain LRVs. The potential CCPs and monitoring
parameter candidates are summarized in the Table 2. We
identified appropriate monitoring parameters for therespective technologies based on simplicity and feasibility ofreal-time monitoring, and on the contribution to pathogen
inactivation.
Regression tree models allowed to identify monitoring
parameters and to estimate the storage time according tostorage conditions. The requisite storage time for pathogen
inactivation in urine and fecal sludge was determined mainly
by temperature, and the CLs of temperature were 14.5 °C and22.5 °C, respectively (Fig. 2(b) and 3(b)). Sanitization isachieved faster in thermal processes such as solar heating,pasteurization, drying, composting, microwaving, andthermophilic digestion (Table 2). Temperature can be a
promising monitoring parameter; thus, appropriate CLs oftemperature should be provided for each process. Persistence
and important biocidal factors varied among the types ofpathogens; therefore, the monitoring parameter and its CL
should be determined using an appropriate surrogate for thepathogen that causes local endemic infectious diseases.
Future studies should investigate the inactivation of phages,
mammalian viruses, and helminth eggs in a wide range oftemperatures and environmental conditions (Table 3).
Sufficient inactivation does not always occur in a single
treatment process; however, it is achieved by combination oftreatment processes. Thermophilic conditions are not always
present in the composting reactors. Pre-stabilization by LAF
before composting realizes sanitization and stabilization in
one month. The use of easily soluble carbohydrates (e.g.,

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4.2 Needs of the inactivation kinetics model
The efficacy of pathogen inactivation is affected by thesurrounding environmental conditions, including climatic
factors and physicochemical properties of the matrices.
Therefore, the storage times required to achieve a certain
LRV, inactivation rate, and decimal reduction time are notcomparable when the properties of the matrices andoperating conditions vary within and among studies. Arriving
at general conclusions regarding the sanitization potential of
an individual technology and comparing it across thetechnologies is difficult because the experimental conditions
are not comparable within or among studies.
The target LRVs were set based on the risk assessment in
the 2006 WHO guidelines. The WHO determines the target
LRVs aiming at 10−6 disability adjusted life years (DALYs),
but it can be changed under a different exposure scenario,
and the estimated storage time is changed accordingly.130 In
addition, the target LRVs were reduced when we aim at 10−4
DALYs, which is a cost-effective target proposed by Mara.131
With the relaxed requirements for LRVs, the technologies weevaluated as “low” can be a stand-alone process for
sanitization. Therefore, evaluations in the Table 2 are reliable
for the predefined specific target LRVs in this study.
An inactivation kinetics model that represents the LRV as
a function of storage time and chemical or physicalindicators is used to estimate LRVs at a certain storage timeand matrix conditions.9 The inactivation kinetics model isalso used to evaluate the sanitization potential acrossresource-recovery technologies under specific conditions.9
When the inactivation profile is defined as a function oftreatment/storage time, estimates of storage time are affectedby the inactivation profiles, that is, straight decay, tailing,shouldering, or combinations. A tailing-off profile derives
estimates of a much longer storage duration compared to afirst-order decay and shouldering profile. The shape of the
kinetic curve and mechanisms behind the profile should be
identified, especially when pathogens remain infective formore than several months. Daily fluctuations in theproperties of the matrices affect the inactivation kinetics

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Table 2 Potential of sanitization, feasible monitoring parameters, and indicator microorganisms in a technology for resource recovery from (A) urineand (B) fecal sludge(A)
Technologies for
urine

Sanitization potential

Indicator
microorganisms

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Storage

Moderate, depending on Phages andstorage temperature and helminth eggs
dilution rate
Solar heating and High, depending on
Phagespasteurization
storage temperature
Nitrification anddistillation
Membranetechnologies

Struviteprecipitation

Alkalidehydration

Low in nitrification, buthigh in distillation
High for hydrolyzed
urine, but low whenurea-hydrolysis isinhibited
Low. Initial heating
prior to drying or drying
at higher temperature
could be effective
High, but moderate forhelminth eggs

PhagesPhages. DNA
viruses/phages
should beinvestigated infuture studies
Helminth eggs

Monitoring parameters

Controllable parameters

Storage time, which can be reduced at>24.5 °C; temperature; dilution ratio;
NH3 concentration
Storage time and temperature. Bacterial
inactivation can be achieved in 1 day at>45 °C

Dilution ratio, storage time,
temperature when heating
devices are available
Dilution ratio, storage time,
temperature when heating
devices are available

Storage time and NH3 concentration fora ssRNA phage. Storage for 1 day causesenough inactivation of Gram-negative
bacteria and a ssRNA phage
Drying temperature

Storage time

Temperature and storage time

Storage time and drying
temperature. Post-storage for 4
d at >42 °C is recommended
for helminth inactivation

Storage time, moisture content
and drying temperature (<55°C)

Ion
No studies
exchange/sorption(B)
Technologies forfecal sludge

Sanitization potential

Indicator
microorganisms Monitoring parameters

Controllable parameters

Storage

Phages andModerate, depending
on storage temperature, helminth eggs
NH3 concentration anduse of desiccants

Storage time; temperature (a criticalvalue is 22.5 °C for helminth
inactivation); pH (>10); and NH3
concentration

Alkalisanitization

High. Bacteria regrowth
should be mitigated by
overdosing
Moderate, depending
on storage temperature

Helminth eggs
and phages

High, depending on
storage temperature
Moderate, dependingon operating
temperature

Helminth eggs

Storage time (several hours to 3 d, 3
weeks for helminth eggs), pH (>10);
and NH3 concentration
Storage time, 6 months (T < 20 °C), 2
months (T < 30 °C), and 0.5 month (T> 30 °C); temperature; NH3concentration; pH (>8.8)
Temperature and storage time (5 d at
>40 °C)Operating time, temperature, C/N ratio
and aeration. 8 weeks composting with
1 month at thermophilic condition(50.7–58.7 °C). Post-storage for 1.5–2
years is recommended for incomplete
composting
Stocking density, pH, composting
duration
Retention time

Ammonia/urea
sanitization

Solar drying andpasteurization
Composting

Vermicomposting Low. Needpost-treatment
Black soldier fly Low. Needcomposting
post-treatment
Lactic acid
Uncertain
fermentation

Helminth eggs
and non-ssRNA
virusesHelminth eggs
and Salmonella

Helminth eggs
and phages
Helminth eggs

1020 | Environ. Sci.: Water Res. Technol., 2023, 9, 1008–1029

For bucket latrines and container,storage time, temperature, pH,ammonia, moisture content; for single
pit latrine and double pit latrine,
storage time, pH, ammonia, moisture
content; for septic tank, storage time;
for twin pits for pour flush toilet,
storage time; for double alternating dry
pit, storage time, pH, ammonia,
moisture content; for double
dehydration vaults, storage time, pH,ammonia, moisture content
Storage time and pH

Storage time and NH3 concentration

Storage time and temperature
Retention time, C/N ratio and moisture
content

Stocking density, composting duration

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Table 2 (continued)(B)
Technologies forfecal sludge

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Anaerobic
digestion
DryingMicrowaving

Sanitization potential
High in thermophilic
digestion, moderate inmesophilic digestion
High
High

Indicator
microorganisms Monitoring parameters

Controllable parameters

PhagesRetention time

Temperature and retention time (30 din thermophilic digestion at >52 °C)

Heating time, temperature
Heating time, temperature
Microwave power level, the amount of Microwave power level, the amount offecal sludge treated and operating time fecal sludge treated and operating timeparameters and can significantly affect the prediction of
microorganisms that survive for more than several months.
Future studies should develop an inactivation kinetics modelthat considers the temporal changes in environmental
conditions over a longer storage period.

4.3 Appropriate surrogate microorganisms
We focused on the inactivation of viruses, phages, bacteria,
and helminth eggs, as they have often been used indisinfection experiments. Helminth eggs, which survived thelongest among the reviewed microorganisms, were effectively
inactivated in treatments that created a thermal
environment, including composting, thermophilic anaerobic
digestion, drying, and pasteurization. Bacterial spores suchas Bacillus cereus and Clostridium perfringens are extremely
resistant to thermal treatment;132 however, we did notconsider them because they are not pathogens. ISO 30500
provides restrictions on the number of viable spores in
products from sanitation systems.12
The target LRVs were highest for phages and viruses
because enteric viruses are excreted in feces in relatively highnumbers and survive in soil and aquatic environments for along period.133 Among the reviewed technologies, phages,
specifically double-stranded DNA phages, are conservative
surrogates for determining CLs in a CCP. Nevertheless, thenumber of reports on virus inactivation is still smaller thanthat on bacteria and helminth eggs. Future studies areneeded to investigate the inactivation of viruses or phages,
and to assess whether they are conservative indicators
compared to helminths and bacteria.

Bacteriophages are often used as surrogates for
mammalian viruses because they are considerably easier tocultivate. Variability in tolerance among viruses andbacteriophages can be explained by the form of the viralgenome (i.e., single-stranded RNA, double-stranded RNA,
single-stranded DNA, and double-stranded DNA) andsensitivity to indigenous microbial activity.35,39 Furthermore,tolerance to disinfection varies among environmental andlaboratory strains in a single serotype.134–136 Virus adaptation
to disinfection can be explained by the dynamic alternationof dominant sequences in a viral population, which isinduced by virus evolution against selection constraints.137
Virus inactivation in a sanitization process (e.g., raisingtemperature, pH, and ammonia concentration) should be
investigated with an insight into virus population genetics;
the expected maximum tolerance of viruses should beconsidered when determining sanitization conditions. As
only few viruses are zoonotic, a consideration about if
reduction of viruses is required in a circular system for nonfood use is necessary.

4.4 Designing a multi-barrier to attain sanitization andstabilization
The sanitization potential of a single technology isdetermined by the biocidal factors inherent to thetechnology,
includingtemperature,pH,ammonia
concentration, moisture content, and indigenous microbialactivity (Table 4). Individual technologies were compared tothe inherent biocidal effect of these factors (Table 4). It
should be noted that susceptibility to biocidal factors differs

Table 3 Knowledge gaps and research priorities in the fate of pathogens

Technologies
For urine
Solar heating andpasteurization
Membrane technology
Ion exchange/sorption
For fecal sludge
Alkali sanitization
Solar drying andpasteurization
Lactic acid fermentation

Types of microorganism and conditions to be examined
Non-ssRNA phages and mammalian viruses, in mesophilic and thermophilic temperatures, with exposure to
sunlight
Helminth eggs, non-ssRNA phages and mammalian viruses, in various temperature conditions
Various types of microorganisms in various temperature conditions
Various types of phages and mammalian viruses, in various temperature conditions
Various types of phages and mammalian viruses, in a mesophilic temperature, with exposure to sunlightHelminth eggs, various types of phages and mammalian viruses in a mesophilic temperatureThis journal is © The Royal Society of Chemistry 2023

Environ. Sci.: Water Res. Technol., 2023, 9, 1008–1029 | 1021

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Table 4 Biocidal factors (+) inherent in the individual technologies, regarding temperature, pH, uncharged ammonia concentration, moisture content
and microbial/enzymatic activity

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Technologies
For urine
Storage
Solar heating and pasteurization
Nitrification and distillation
Membrane technologies
Struvite precipitation
Alkali dehydration
Ion exchange/sorption
For fecal sludge
Storage- Bucket latrine and container- Single pit latrine- Septic tank
- Twin pits for pour flush toilet- Double alternating dry pit- Double dehydration vaults
Alkali sanitization
Ammonia/urea sanitization
Solar drying and pasteurization
Composting
Vermicomposting
Black soldier fly composting
Lactic acid fermentation
Anaerobic digestion
DryingMicrowavingaHigh temperature

Alkali/acid pH

Uncharged ammonia

Desiccation

Microbial/enzymatic activity

−
+
+
−
−
−
−

+
+a
−
+a
−
+
−

+
+a
−
+a
−
−
−

−
−
−
−
+
+
−

+
−
+
−
−
−
−

−
−
−
−
−
−
−
−
+
+
−
−
−
+
+
+

+
+
−
+
+
+
+
+
−
−
−
−
+
+
−
−

+
+
−
+
+
−
+
+
−
−
−
−
−
−
−

−
−
−
−
+
+
−
−
+
+
−
−
−
−
+
+

+
+
+
+
+
+
−
−
−
+
+
+
+
+
−
−

Not significant (−) when urea-hydrolysis is inhibited.

among microorganisms. For example, Salmonella spp. wererapidly inactivated at pH 12 in ammonia- and carbonate-freebuffer solutions, whereas Ascaris eggs and spores of
Clostridium
perfringens
were
notinactivated.121,138
Temperature, pH, ammonia concentration, and moisture
content were used as monitoring parameters. Continuous
monitoring of microbial and enzymatic activities is notconsidered practical because culture methods usually requirean overnight incubation period. Indicators that represent theperformance of biological treatments (e.g., C/N ratio andodor) could be alternative monitoring parameters.
The operational conditions of individual technologies canbe optimized for sufficient pathogen inactivation bycontrolling temperature, pH, ammonia concentration, andmoisture content; however, they are not always controllable.
We have distinguished the controllable parameters from themonitoring parameters in the Table 2. For example, a higher
temperature is favorable for rapid inactivation, but thetemperature inside a single pit latrine is merely changed
owing to its large capacity. Meanwhile, smaller containers(e.g., buckets latrine) can be moved to sunlight to promote
pathogen inactivation by increasing the temperature of thematrix. When biocidal factors cannot be intensified using acertain technology, greater inactivation is achieved byextending storage time.
This study investigated the operating conditions, with afocus on the pathogen inactivation performance of sanitation
technologies. Nevertheless, we must emphasize that1022 | Environ. Sci.: Water Res. Technol., 2023, 9, 1008–1029

appropriate sanitization conditions do not always lead tostabilization. We identified resource recovery technologies
that are not stand-alone processes for sanitization.
Specifically, pathogens can remain infective during biologicaltreatments without generating heat, such as nitrification,
vermicomposting, BSF composting, and LAF. Increasing theoperating temperature for pathogen inactivation affects
valuable microorganisms involved in the organic matterstabilization. Similarly, ammonia treatment, drying, andthermophilic storage result in partial stabilization of organic
matter because they hamper microbial activity in biologicaltreatments.139 Liming is an effective disinfection method thatis applicable for on-site treatments before manual emptying;
however, an extremely alkaline pH inhibit indigenous
microbial activity.140 Therefore, stabilization and sanitization
may not be attained simultaneously, and the CL value should
be set so as not to hinder biological treatment.
A multibarrier approach should be designed to attainstabilization and sanitization with an appropriate
combination of technical barriers. To achieve this, werecommend that the pathogen inactivation efficiency of every
technology be evaluated in advance under the optimum
conditions for stabilization. An inactivation kinetics model
for pathogens should be developed for individual
technologies and used to estimate the level of pathogens
under certain operating conditions. Additional sanitization
processes can be introduced as long as they do not hinder
the stabilization.

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WHO recommends a combination of treatment
technologies and measures other than treatments, including
restrictions on the fertilization of edible plants, use of
personal protection equipment (PPE), and farmland
application techniques.6 We suggest that these technical andnon-technical barriers be introduced and monitored aspriorities when the target LRV is not achieved in a preceding
multiple barrier treatment. Estimation of the LRV in resource
recovery technology is important to provide a clear rationale
for the intensive monitoring of non-technical barriers andbarriers other than treatment technologies.

4 Ellen MacArthur Foundation, Towards the circular economy:
opportunities for the consumer goods sector, 2013.
5 R. Harder, R. Wielemaker, T. A. Larsen, G. Zeeman and G.
Öberg, Recycling nutrients contained in human excreta toagriculture: Pathways, processes, and products, Crit. Rev.
Environ. Sci. Technol., 2019, 49(8), 695–743.
6 WHO, WHO guidelines for the safe use of wastewater, excreta
and greywater, Geneva, 2006, vol. 4 Excreta and greywater
use in agriculture.
7 WHO, Sanitation safety planning: manual for safe use anddisposal of wastewater, greywater and excreta [Internet],
Geneva, 2015, [cited 2019 Feb 4], Available from: https://apps.who.int/iris/handle/10665/171753.
8 US FDA, HACCP Principles and Application Guidelines[Internet], Silver Spring, Maryland, 1997, [cited 2020 Jan 3],
Available from: https://www.fda.gov/food/hazard-analysiscritical-control-point-haccp/haccp-principles-applicationguidelines.
9 W. Oishi, S. S. Kadoya, O. Nishimura, J. B. Rose and D.
Sano, Hierarchical Bayesian modeling for predictive
environmental microbiology toward a safe use of humanexcreta: Systematic review and meta-analysis, J. Environ.
Manage., 2021, 284(February), 112088, DOI: 10.1016/j.
jenvman.2021.112088.
10 Ministry of Health, Decision No. 08/2005/QD-BYT, regarding
issuing the sector standards: Hygiene standards for various
types of latrines, Hanoi, Vietnum, 2005.
11 Ministry of Health of the People's Republic of China,
Hygienic requirements for harmless disposal of night soil,
China, 2012.
12 ISO, Non-sewered sanitation systems — Prefabricatedintegrated treatment units — General safety andperformance requirements for design and testing(Reference number: ISO 30500:2018(E)), 2018.
13 ISO, Faecal sludge treatment units — Energy independent,
prefabricated, community-scale, resource recovery units —Safety and performance requirements (Reference number
ISO 31800:2020(E)), 2020.
14 J. McConville, C. Niwagaba, A. Nordin, M. Ahlström, V.
Namboozo and M. Kiffe, Guide to sanitation resourcerecovery products and technologies: A supplement to thecompendium of sanitation systems and technologies, Swedish
University of Agricultural Sciences (SLU), Uppsala, 1st edn,
2020.
15 C. Schönning, T. Westrell, T. A. Stenström, K. ArnbjergNielsen, A. B. Hasling and L. Høibye, et al., Microbial risk
assessment of local handling and use of human faeces,
J. Water Health, 2007, 5(1), 117–128.
16 Sanitation and Disease in the 21st Century: Health andMicrobiological Aspects of Excreta and Wastewater
Management [Internet], 2019, Available from: https://www.
waterpathogens.org/toc.
17 W. Oishi, B. Vinnerås, J. B. Rose and D. Sano, Predictive
environmental microbiology for safe use of sanitation
products in agriculture: Challenges and perspectives,
Environ. Sci. Technol. Lett., 2021, 8(11), 924–931.

4.5 The limitations of this review
This review has two limitations. First, greenhouse gas
emissions were not considered. We describe that long storage
or retention times are necessary for disinfection; however,greenhouse gas emissions from fecal sludge can increasesignificantly with increased storage time.141,142 Reid et al.
demonstrated that replacement to a low-CH4 emission
technologies (e.g., composting toilet) yields co-benefits forboth greenhouse gas mitigation and water and sanitation
development.141 Second, some novel thermal treatment
systems are not included that have gained traction in recent
years, e.g., Omni Processors.143 These technologies may
completely inactivate pathogens and produce a product thatcan be used for agriculture (e.g., biochar).

Conflicts of interest
There are no conflicts of interest to declare.

Acknowledgements
This work was supported by the Research Institute for
Humanity and Nature (RIHN: a constituent member of NIHU)
under Grant No. 14200107 and JSPS KAKENHI under Grant
No. 20J10868 and 22K20442.

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