A review on the use and applications of Volatile Fatty Acids on fecal sludge sanitization

Adequate sanitation is an important basis for a healthy and dignified life, economic development, and sustainable use of the resources from the environment (Bartram & Cairncross 2010). In developing countries, fatalities from diarrheal diseases account for 88% and this is mainly caused due to the lack of full access to sanitation, poor hygiene, and the use of unsafe drinking water. Improved sanitation can play a greater role in reducing the incidence of death which is caused by diarrhoea (Strande et al. 2014). As of 2015, 2.4 billion people were still without access to proper sanitation. This figure comprises 892 million people who practice open defecation, especially in rural areas. Even though improved sanitation facilities have shown less progress, it has increased from 59% in 2000 to 68% in 2015 (WHO/UNICEF 2015).

Due to urbanization and the increase in population growth and climate change freshwater resource scarcity can be exacerbated. Human feces have a substantially higher amount of bacteria, helminths, and viruses compared to urine. As a result of the huge number of pathogens present, feces must always be treated appropriately. Poor sanitation systems for human excreta may contribute to disease transmission when used as fertilizer in agricultural fields (Bashir et al. 2020).

A study conducted by Jensen et al. (2005) revealed that the application of partially composted fecal wastes from latrines has caused 30% of hookworm infections among the local populations in Vietnam. Therefore, proper management of human excreta should be conducted for disposal and treatment. Fecal sludge management (FSM) is among the most significant global challenges. The treatment methods used for fecal sludge (FS) must be environmentally friendly, cost-effective, and should be able to use in agricultural fields to enhance the soil nutrients (Zewde et al. 2021).

The emerging economies in developing countries are highly reliant on on-site treatment like pit latrines and septic tanks. The need for a safe and sustainable sanitation system is significant given that 2.7 billion people rely on on-site sanitation today, with that figure predicted to climb to 5 billion by 2030 (Strande 2014). Once the septic tanks or pit latrines are filled, much of the sludge that is accumulated is directly discharged without any treatment into any open drains, open lands, irrigation fields, and sources of surface water. As a result, its disposal poses a major risk to public health (Ingallinella et al. 2002).

Inadequate and unsafe disposal of human excrement can contaminate ground and water sources, as well as provide breeding grounds for flies and mosquitos that can transmit diseases. Feces may attract domestic animals and vermin, which can both increase the potential for disease. It can also create an unpleasant environment in terms of odour and sight. The introduction of safe excreta disposal can reduce the increase in intestinal and helminth infections. Excreta-related communicable diseases include cholera, typhoid, dysentery, diarrhoea, hookworm, schistosomiasis, and filariasis.

Therefore, to address this issue different methods have been used to sanitize FS. Short-chain fatty acids with two to six carbon atoms are referred to as volatile fatty acids (VFAs). VFAs can be produced from food waste through anaerobic digestion (AD) (Kim et al. 2006). There are various kinds of VFAs, each with its own set of properties and uses. These are the acetic, propionic, and butyric acids and Table 1 summarises their chemical structure and applications (Zigova & Šturdík 2000; Bhatia & Yang 2017). AD has also been demonstrated to be a feasible and effective treatment for food waste. Food waste is a suitable substrate for AD since it contains a lot of moisture and organic materials. Solubilization, hydrolysis, acidogenesis, and methanogenesis are four major processes in anaerobic fermentation that can produce VFAs and methane continuously (Leng et al. 2018; Wang et al. 2018).

Table 1

Characteristics of VFAs

VFAs .	Chemical formula .	Usage/applications .	References .
Acetic acid		Food additive, solvent, vinegar, ester production, chemicals, vinyl acetate monomer (polymers, adhesives, dyes)	Bhatia & Yang (2017)
Butyric acid		Animal and human food additive, chemical intermediate, solvent, flavouring agent	Zigova & Šturdík (2000)
Propionic acid		Esters used food industry as aroma additive, food additive, flavouring, pharmaceuticals, animal feed supplement, fishing bait additive	Cheryan (2009)

VFAs .	Chemical formula .	Usage/applications .	References .
Acetic acid		Food additive, solvent, vinegar, ester production, chemicals, vinyl acetate monomer (polymers, adhesives, dyes)	Bhatia & Yang (2017)
Butyric acid		Animal and human food additive, chemical intermediate, solvent, flavouring agent	Zigova & Šturdík (2000)
Propionic acid		Esters used food industry as aroma additive, food additive, flavouring, pharmaceuticals, animal feed supplement, fishing bait additive	Cheryan (2009)

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The acid-forming bacteria, which include acetogenic and homoacetogenic bacteria, are the microorganisms that are primarily responsible for VFA synthesis (Xu et al. 2018). Due to its better value and a broader range of applications the production of VFAs is getting attention. The type of substrate used for the production of VFAs has an impact on the amount and composition of the VFA produced (Atasoy et al. 2018). This is because the characteristics of the substrate determine the degree of acidity and the prevalent metabolic functions during the fermentation process (Chen et al. 2013).

Since food wastes have high volatile solids and water content, they are considered a primary source of decomposition, hazardous gas emissions, and groundwater pollution. Because of their higher organic content and biodegradability food wastes can generate energy. Acidogenesis-derived VFA can be utilized as a source of energy and carbon for biological nitrogen elimination (Ahammad et al. 2008) and biodegradable polyhydroxyalkanoate (PHA) storage (Lim et al. 2008). Various methods have been also applied for pathogen inactivation in FS such as, the application of lactic acid fermentation, lime, and urea. These treatment techniques require widely available materials: molasses, hydrated lime, and urea to effectively sanitize FS.

This review paper attempts to close the gap by analysing the current research developments and problems with FSM technologies. The key emphasis is on the challenges related to the treatment and disposal of FSM in the development of urban centres, where there is a clear need for improvement in various treatment methods. The methodology designed in this article presents an orderly and in-depth assessment of FSM's significant challenge, collects the current state of research on various FS treatment technologies, and assesses the advantages of FS as soil modification via the various treatment technologies.

Due to the vast range of characteristics and stability of FS, the treatment method is difficult, as it drives the selection of technological solutions (Sonko et al. 2014; Gold et al. 2016). The management of FS needs urgent measures, including collection, transport, treatment, disposal, and application of the end use of FS in various sectors, such as bio-gas extraction, soil amendment, and combustible or dry fuel (Strande et al. 2014). Treatment technologies for FS are mainly based on technologies designed for the treatment of wastewater, but the characteristics of FS are different from wastewater technologies used for wastewater that cannot be directly used for FS. Therefore, the efficiency of the treatment method will be less (Do & Bui 2018).

During sludge processing, pathogens are inactivated by different mechanisms. The major mechanisms that lead to pathogen destruction in four common sludge management technologies are sludge digesters, sludge drying beds, composting systems, and land application systems. The effectiveness of each of these mechanisms is determined by a variety of environmental design and operational factors. Temperature and treatment time have been recognized as possibly the two most essential parameters in attaining considerable pathogen elimination during sludge management. Other methods for pathogen elimination during sludge treatment include desiccation, raising pH, and increasing free (unionized) ammonia (NH₃) concentration (Sahlström 2003). Biological stabilization techniques can lower the volatile organic portion of sludge, limiting disease vector attraction and bacterial growth due to the loss of available organic substrate (Zamri et al. 2021).

Many FS treatment technologies are mainly established to be used to treat wastewater and wastewater sludge. Since the characteristics of FS differ substantially from those of wastewater, these treatment technologies cannot be directly applied to FS because they will have a significant impact on the treatment efficiency (Lim et al. 2008; Mramba et al. 2020). As a result of improper FS disposal methods, many communities in low- and middle-income countries are currently suffering comparable concerns of health prevalence and environmental contamination. FS and wastewater compositions can vary greatly between countries and contexts, driven by differences in user practices, water usage and consumption, climate, population density, area limitations, and accessibility. This is an important consideration in selecting product/s and their likely success in treating FS and wastewater.

Due to the enormous concerns and difficulties associated with FS management, a wide range of technical, financial, and institutional approaches are needed to address the situation. Locally available technology should be adopted in low- and middle-income countries to improve FSM. Those management approaches will be sustainable if they are eco-friendly, socio-economically feasible, and acceptable. The efficiency of various treatment strategies widely used to reduce pathogen inactivation in FS varies. Several FS treatment techniques for agricultural reuse have recently been implemented, including VFAs, lactic acid, lime, yeast, and ammonia pre-treatments. The authors emphasize the effect of VFA on pathogen inactivation because VFAs can be produced from readily available food wastes and can be considered a low-cost sanitizing method for low-income countries.

VFAs are carboxylic acids with two to six carbon atoms that have a low molecular weight. VFAs are now largely generated utilizing petrochemicals in conventional chemical methods (such as methanol carbonylation, propane oxidation, and butyraldehyde oxidation) (Atasoy et al. 2018). However, these methods are highly energy-intensive and depend on a non-renewable source, which makes them unattractive from an economic and environmental perspective. It has been reported that greenhouse gas (GHG) emissions from the production of acetic acids in the petrochemical industry emitted 3.3 t-CO₂ eq/t from the cradle to the grave. Therefore, VFA production by biological pathways from organic waste is gaining more attention because of its economic and environmental advantages (Ramos-Suarez et al. 2021).

VFAs derived from food wastes have the potential to be effective electron donors for the removal of nitrogen and phosphorus (Sangkharak & Prasertsan 2013; Van Thuoc et al. 2019). VFAs are platform chemicals used in a broad range of industries, including agriculture, food, pharmaceutical, and chemical. VFAs are predominantly produced through chemical synthesis from petroleum-based feedstocks. This process typically involves high temperatures, high pressures, and catalysts for conversion to VFAs (Dionisi & Silva 2016). Acetic, propionic, and butyric acids are produced from the acidogenesis process of organic matter. The type of substrate, organic loading rate, hydraulic retention time (HRT), and other operational conditions all have an impact on the determination of the composition of VFAs (Alkarimiah et al. 2011; Aris et al. 2017). AD provides an effective approach to the treatment of human waste (Cai et al. 2016). It allows the treatment of organic waste by avoiding emissions of volatile organic compounds, stabilization of organic matter, and accumulation of effluent with good fertilizing qualities in addition to energy recovery through methane (Cai et al. 2018). Some studies, however, have reported inadequate inactivation of pathogens in AD (Cheng et al. 2020).

Fundamentally, AD is a biological process where organic matter (carbohydrates, lipids, and proteins), except for the lignin components, is degraded in the absence of oxygen, generating methane and carbon dioxide (Huang et al. 2016). The main processes in AD are hydrolysis, acetogenesis, acidogenesis, and methanogenesis. When treating solids-rich waste streams, hydrolysis is the step that defines the rate with pH, temperature, C/N ratio, and HRT, which have been identified as the key factors for VFA formation and its conversion to methane (Yu et al. 2016; Zhou et al. 2020).

Waste-activated sludge (WAS), a major by-product of sewage treatment plants (STPs), has raised serious concerns due to its extensive production, significant environmental risks, and economic burden. The anaerobic fermentation process used in WAS is commonly related to a variety of environmental factors. For instance, the optimal concentration of surfactants could effectively improve short chain fatty acid (SCFA) production (271.4–472.7%) (Luo et al. 2019). Surfactants and nanoparticles (NPs) are the two typical pollutants in WAS. Different effects from each pollutant are expected to impact the subsequent WAS treatment. A study conducted by Luo (Luo et al. 2022) revealed that CeO2 NPs coexisting with surfactants had antagonistic effects on SCFA production (10.7 and 33.9% suppression by hexadecyl trimethyl ammonium bromide (HTAB) and sodium dodecyl benzene sulfonate (SDBS), respectively). As a widely used antibacterial agent, para-chloro-meta-xylenol (PCMX) showed dose-dependent effects on sludge fermentation for VFAs. The presence of PCMX at environmental levels (10–20 mg/g total suspended solids (TSS)) promoted the VFA production by 2.5–3.0 folds but caused evident inhibition at excessive addition (Du et al. 2022)

VFAs are usually produced during the hydrolysis/acidification phase of AD. To produce VFAs from food wastes via the AD, the last stage, methane production must be inhibited. Several studies have shown that 2-bromoethane sulfonic acid (BES) has the capacity to inhibit the activity of the methanogens (Chidthaisong & Conrad 2000), with an optimal concentration of around 50 mM for complete inhibition. Another investigation revealed that the application of 1 mol/mL of BES could inhibit the activity of methanogens by 60%, increasing the build-up of acetate (Mohan et al. 2008).

AD is extensively used to treat bio-wastes such as food waste, municipal solid waste, and sewage sludge. Moreover, it can provide energy recovery, stabilize waste, recycle nutrients to be used as fertilizers, and can reduce GHG emissions. Pathogens are exposed to complex environments during AD (Salsali et al. 2008; Jiang et al. 2020). Various factors may affect the effectiveness of pathogen inactivation, including pathogen types, operational conditions, intermediates produced, and the substrates used. These factors can determine the amount of energy produced, and optimizations are required to achieve mutual benefit from energy recovery and pathogen inactivation (Jiang et al. 2018).

In general, to achieve sustainability in sanitation, it is important to remove pathogens from FS. The main objective of disposal is to protect the environment and to increase the safety of nutrient reuse from human excreta as fertilizer (Kunte et al. 2000a). The different types of pathogens in FS include Vibrio cholera, Salmonella paratyphi, Shigella spp., and Salmonella typhi from bacteria (Riungu et al. 2018b). The common viruses are Rotavirus, enteric adenovirus, and norovirus and the protozoa include Entamoeba histolytica, Cryptosporidium hominis, and Giardia intestinalis. Additionally, the most common helminth eggs are those originating from Ascaris lumbricoides and Trichuris trichiura (Riungu et al. 2018a).

Some studies have shown that acidity and alkalinity can significantly inhibit the growth of pathogens. The success of acidification often depends on many operational factors in the process. Process disruptions and deterioration in VFA production can be explained by factors such as the presence of toxic substances and microbial competition from acetogens and methanogens. Recently, the factors affecting competition from acetogens and methanogens have been the subject of much research, as the removal of undesirable methanogens from plants on a large scale was complicated and effective control strategies preventing their growth are only beginning to be developed, including the substrate, pH, temperature, HRT, and a few other factors (Popat et al. 2010; Rojas-Oropeza et al. 2017).

The pH of sludge is an important feature since it can affect the distribution between unionized and ionized forms of organic acids. In the anaerobic fermentation process, pH is an essential factor because pH in the environment can affect the metabolic pathways by affecting the activity and stability of intracellular enzymes and modifying the selective permeability of cell membranes and the toxicity of substrates in the environment (Daneshmand et al. 2012; Munasinghe-Arachchige et al. 2019). Generally, methanogens have an approximate value of pH in the range of 6.6–7.5 and the process of methane formation will be suppressed when the pH is above eight or below six.

In contrast, they are more adaptable to pH and less sensitive to changes in pH in the environment (García-Fernández et al. 2019). Researches have confirmed that acetogens can still be subjected to anaerobic fermentation even under conditions of acidity and alkalinity accompanied by the accumulation of VFA. During anaerobic fermentation, the accumulation of VFA will cause the pH to start to drop and affect the living environment of the methanogens (Rothrock et al. 2017). However, this condition can be avoided within a certain range due to the buffering effect of the digestive juices. Normally, alkaline fermentation technology is widely used to improve acid production. To summarize, though the pH has an impact on hydrolysed bacteria, acetogens, and methanogens the effect of pH on the metabolic efficiency of different processes is different. Therefore, the use of pH to regulate the production of acid from the fermentation of the sludge is of great importance (Giannakis et al. 2017).

Temperature affects the composition of the cell membrane. Cell components such as proteins, lipids, and carbohydrates are responsible for cell transport phenomena and can withstand a narrow temperature range. An increase in temperature beyond the usual membrane temperature may result in a change in the molecular structure of the membrane (Odey et al. 2019). Cell membrane fluid may increase, allowing organic acids to diffuse more rapidly into the cytoplasm (Kim & Ndegwa 2018).

It was found that the decrease of Salmonella content in the digestive wastewater of a digester at dosing with volatile organic acids depends on pH, temperature, the length of the acid chain, as well as the concentration and the composition of acids present. At mesophilic temperatures, acidic pH results in stronger Salmonella inhibition, whereas at higher temperatures, a neutral pH is more inhibitory. It has been suggested that acid-phase reactors that operate at higher temperatures and a lower pH can attain a significant reduction in Salmonella spp. (Jin et al. 2018).

Different kinds of sewage sludge could cultivate discriminate microbial communities; therefore compositions of substrates are important for the production of VFA. Existing studies have shown that a substrate with a high solid content can cause an ammonium press, which can direct the structure of the bacterial community towards strengthening the syntrophic acetate oxidation reaction (Leng et al. 2018). On the other hand, in some other studies, it was shown that a substrate that is rich in protein content may limit propionate production. Acetogens can also oxidize VFA (C > 2) to acetate and hydrogen, a process that requires low partial pressure H₂ to become thermodynamically favourable (Strazzera et al. 2018).

In a single-stage process, low hydrogen levels are provided by the activity of hydrogenotrophic methanogens. In the reactor for hydrolysis, which does not possess nor has low methanogenic activity, hydrogen can potentially be both produced and consumed by different groups of acetogens. Knowledge of acid production is, in itself, quite good, since acids have been studied in many different processes, both for individual compounds and complex materials and for individual and mixed cultures of microorganisms (Atasoy et al. 2018; Strazzera et al. 2018). However, less is known about how to direct the process towards the production of specific VFA from a matrix of complex substrates, e.g., food waste or sewage sludge, due to the complexity of AD and a large number of different possible routes. Table 2 depicts the VFAs produced from different streams.

During the AD process besides the effects of pH and VFA produced, the concentrations of the feed substrate can also determine bacterial survival. The toxicity of VFA is related to the dissociation of the acid molecule: non-dissociated-VFAs are capable of passing through the cell membrane of microbes through passive diffusion and will dissociate by disrupting internal pH, affecting the tertiary level of protein structure and inhibitory growth of microorganisms (Riungu et al. n.d.). Besides, the non-dissociated-VFAs can make the cell membrane porous, which enables the leaching out of cell contents and disintegration of microorganisms. The antibacterial effectiveness of non-dissociated VFA has been studied in the treatment of Escherichia coli infections in rabbits and pigs and reported that at higher pH, less non-dissociated VFA was found to inactivate pathogens (Zeng et al. 2019).

Differences in the ability to remove E. coli under different conditions were observed in an experiment conducted by Ziemba and Peccia (2011). First, log removal of E. coli at a high temperature of 35°C was much better than log removal at room temperature of 23°C for all samples. In addition, log removal in mixed market waste (MMW) is comparable to log removal in samples with VFA added and, in fact, higher than log removal in samples containing acetic acid, propionic acid and butyric acid at a concentration of 2,000 mg/L. At the same concentrations, fecal samples containing acetic acid, propionic acid, and butyric acid showed similar log removal of E. coli at VFA concentrations of 3,000 and 4,000 mg/L. However, at a concentration of 2,000 mg/L, acetic acid was added to the sample and the order of removal (acetic acid > propionic acid > butyric acid) showed the highest rate of E. coli log removal. Finally, at a low pH of 4.8, there is more E. coli removal in the feces than at a normal pH of 6.2 (Jiang et al. 2020). Table 3 shows the pathogens that are excreted in feces and the related disease symptoms.

AD in a mesophilic medium (35–40 °C) is the most widely used method for stabilizing primary and secondary sludge in municipal wastewater. However, many types of pathogens can still thrive in such treatments (Fontana et al. 2020; Xu et al. 2020) and high temperatures are often required to improve sludge stabilization rates and promote pathogen reduction. During the AD process, pathogens are also subjected to higher concentrations of organic acids. The efficacy of organic acids depends on concentration, pH, temperature, exposure time, and the degree of susceptibility of certain types of pathogens (Villagrán-de la Mora et al. 2019).

These factors, alone or in combination, may affect microbial injury in AD. The degree of damage will probably vary and be a function of the susceptibility of a particular microorganism. Temperature affects the composition of the cell membrane. Components of the cell, such as proteins, lipids, and carbohydrates, are responsible for the phenomenon of transport within the cell and can tolerate a narrow range of temperatures (Lacey et al. 2018). When the temperature rises above the temperature to which the cell membrane is accustomed, the molecular structure of the cell membrane can be altered. It may increase the fluidity of the cell membrane, which enables more rapid diffusion of organic acids into the cytoplasm (El Kadri et al. 2020).

Clostridium Perfringens is an anaerobic, Gram-positive, spore-forming, and heat-resistant bacterium. Since these types of organisms are found in large quantities in municipal sludges, they are considered an indicator of the survival of pathogens in the process of sludge treatment. However, fewer studies have been conducted to evaluate the probable fate of C. perfringens during the AD process. Several studies have found that acid-phase digestion at mesophilic temperatures reduces pathogens slightly more than mesophilic digestion. This is probably due to the inhibitory effect of organic acids (Rood et al. 2018). A study conducted by Taherzadeh et al. (1997) revealed that at pH 5.5, a typical pH value for acid-stage digesters, there was no significant change in the concentration of C. perfringens in the temperature range from 35 to 55 °C. However, at pH 4.5, the inhibition of C. perfringens increased as the temperature increased. This may be due to the protonation of organic acids at this pH range, which is generally considered to be more toxic to C. perfringens than the dissociated acids (Rood et al. 2018).

Treatment with acid at high temperatures has considerably reduced the concentration of C. perfringens. However, the extent of reduction varied depending on the organic acid concentration, temperature, and pH. Treatment with acid at high temperatures resulted in a much greater reduction of C. perfringens concentration than at mesophilic temperature. The results obtained suggest that high concentrations of organic acids at pH values of 4.5–5.5 during thermophilic fermentation significantly reduce the concentration of C. perfringens in municipal sludge. It has been observed that the large decrease in the C. perfringens population during the treatment processes was capable of reducing the number of other bacteria by an equal or larger amount (Kiu & Hall 2018). When the concentration of C. perfringens was exposed to either 750 or 1,500 mg/L of VFA, it did not show a significant change. When 3,000 and 6,000 mg/L of VFA concentration has been used the concentrations of C. perfringens were reduced by approximately 18% compared to the initial concentrations (Salsali et al. 2008).

Helminth eggs are considered one of the main biological risks when FSs are directly applied as a fertilizer on agricultural fields to enhance the production rate of crops. Helminth eggs can survive for longer periods than other microbes for instance, 1–2 months in crops and many months in soil, or also it may be viable for several years in FS (Jiang et al. 2020). Helminth eggs are tolerant to different environmental conditions, depending on the composition of the eggshell. Each type of helminth egg has from three to four layers with different physical and chemical characteristics. They have a very strong shell and this helps the egg to survive for much longer periods in FS (Rojas-Oropeza et al. 2017).

Helminth ova contain a shell that has three layers, namely, a lipoidal inner layer, a chitinous middle layer, and an outer proteinic layer. The main advantage of these layers is to give tolerance to the egg during unfavourable environmental conditions. The size range of the helminth eggs that are used for irrigation purposes is between 20 and 80 mm with a relative density of 1.06–1.15. VFA is an intermediate that is produced by the AD process in a two-phase reactor (acidogenic/methanogenic phase), high VFA concentrations can be obtained in the first reactor. The VFAs produced in a two-phase reactor could affect the viability of the pathogens and parasites. Therefore, it is important to determine the impact of VFA in the removal of helminth ova under different temperature conditions. The life cycles and ova morphologies of Ascaris suum and A. lumbricoides are similar (Zhao & Liu 2019).

Lepesteur (2020) conducted a study on A. suum ova to determine the influence of four mixtures of VFA, under mesophilic temperature (35 °C) anaerobic conditions, on the survival rate of the infective larvae. From the results obtained, it was revealed that the presence of acetic and peracetic acids had inactivated bacteria and helminth ova efficiently. Besides this, the combination of VFA can be also used as a disinfectant for pathogen inactivation (Ghiglietti et al. 1995).

Few studies have been conducted to determine the processes by which acids affect the survival of helminth eggs. Sulphuric acid, perchloric acid, peracetic acid, acetic acid, propionic acid, isobutyric acid, n-butyric acid, and isovaleric acid have been used to suppress pathogens in FS. Niz et al (2020) found that when peracetic acid was used as a dispersant, peracetic acid denatured proteins. They observed that this may be related to their ability to make Helminth eggs inactivated by peracetic acid were darker in colour and exhibited an unusual granulation and an oval shape, which may be associated with cell wall damage.

Previous studies have shown that the effect of fatty acids on the viability of A. suum eggs can be affected by acid concentrations and the temperature at which the exposure time occurred. Higher concentrations of acid and higher temperatures can minimize the time required to complete inactivation at 37 °C, and the time required for 1.5 M 100% of eggs treated with pentanoic or hexanoic acids died in less than 10 min (Markou et al. 2018). Previous studies mainly tested the inactivation ability of single fatty acids at high concentrations that are not being produced in the current pilot toilet system.

Therefore, this study was designed to determine the realistic concentrations of fatty acids at the levels produced by the toilet system. Impacts were examined and the levels produced appear to be fully sufficient to inactivate the eggs at least when the eggs were present for 48 h in water containing a solution of acids. As a result, fatty acids and various acid combinations with low concentrations were tested against A. suum eggs in this study. The results of our research showed that fatty acids in certain combinations have effective inactivation ability even at the lower concentrations (Ghiglietti et al. 1995).

An Ascaris egg contains a large amount of fat stored in the form of energy, the initial concentration of which is reduced to 66% during embryonic development. These fats, consisting of VFAs (acetic, butyric, and pentanoic), coincide with the types of acids that adult females release as fermentation products. Thus, it is very likely that some of these acids can be incorporated and used in the egg metabolism process, leading to the hypothesis that the cell walls of a helminth egg can be permeable to these types of compounds. Although the exact mechanism of acid attack on the egg has yet to be determined, this potential permeability would explain the effect of VFAs in reducing the concentration of a viable helminth egg in sludge (Boyko & Brygadyrenko 2017).

The concentration of certain types of organic acids in the environment, membrane permeability, and their interaction with other physical and chemical factors can decrease the viability of helminth eggs. It can be assumed that organic acids can penetrate the egg walls as bacteria do, given that the lipid layer is semi-permeable, and that this permeability varies depending on both environmental conditions and the development stage of the ovum. Once the organic acids enter inside, they will dissociate and accumulate like protons and anions, which leads to the inactivation of the organism (Ray & Sandine 2019).

Presently, urine, black water, and compost from feces and struvites seem to be the most essential new fertilizers obtained from source-separated wastewater systems. Field systems that separate grey water and FS/toilet water (without separating urine and feces) are among the most promising nutrient recycling concepts, as they can be adapted from traditional sanitary systems and their social acceptability is usually higher than the dry sanitation systems. Pathogens are known to be one of the main limitations in the use of wastewater in agriculture and since FS can be highly contaminated, this is a key factor in the introduction of sewerage systems aiming for reusing these wastewaters (Romero et al. 2011).

Species of bacteria, protozoa, helminths, and viruses are among the pathogens of concern in FS. Although these groups occur in high concentrations, published data on the inactivation of enteric viruses in latrine feces, septic tank precipitation, and dry toilet feces are almost non-existent, mainly due to the cost and complexity of viral monitoring procedures due to the complexity of those matrices. Human rotavirus and adenovirus are commonly found in wastewater. They are important aetiological agents in many human diseases, and adenoviruses are widely used as viral indicators of contaminated fecal water because of their high environmental stability. Besides this, adenovirus can survive in different treatment processes therefore they are considered one of the most conservative viruses (Magri et al. 2015).

Generally, the inactivation of viruses and bacteriophages was faster in treatment with higher urine concentration or urea dose, higher pH, and higher temperature than in treatment with low urine/urea content, low pH, and low temperature. Viruses and bacteriophages were inactivated faster in pH 9 buffers than in pH 7 buffers. However, inactivation at pH 9 was slower in almost all cases than in ammonia treatment. At pH 9 at 33 °C, an initial decrease in virus viability occurred, which was more pronounced for AdV than for ReV. The decrease in the viability of the virus was more than 10 times higher than at the same temperature and with higher pH and NH₃ content (Harakeh & Butler 1984).

The inactivation rates k for ReV and AdV showed an increase with the increase of NH₃ concentrations. Moreover, the correlation between pH and inactivation rates is very strong. Therefore, because of their relationship, it is difficult to distinguish between the effects of pH and ammonia, but we can detect both effects at most temperatures. The pH 9 control had a higher inactivation rate than the pH 7 control, so pH affected the inactivation. In the case of AdV, the treatment with 0.15% urea and pH 8.6 at 28 °C had a lower inactivation rate than the pH 9 controls, despite having a considerably low concentration of ammonia, which could be explained by the lower pH of the treatment (Magri et al. 2015).

The inactivation rate of the Reovirus type 3 found at pH 9.5 and 287 mM NH₃ was approximately 65% higher compared with the ammonia-free buffer at pH 9.5. From the study, it can be concluded that the inactivation is partly due to ammonia and partly due to high pH. This strongly indicates that both pH and NH₃ have an inactivation effect on ReV. Similar trends could be observed for the inactivation rates of phages (Romero et al. 2011). Higher inactivation rates were correlated to both higher NH₃ and higher pH values. More studies with ammonia treatment and pH controls with higher pH values are required to separate the impact of ammonia from the impact of pH. Some recent findings described the kinetics and mechanisms of inactivation of the single-stranded RNA phage MS2 under temperature, pH, and NH₃ conditions. The authors found that MS2 inactivation was mainly controlled by the activity of NH₃ over a pH range of 7.0–9.0, while the effect of pH 9.5 was similar to the effect of 43 mM NH₃. They also verified that other bases (e.g., bicarbonate, carbonate) additionally have contributed to the reduction of infectivity of MS2 (Rutjes et al. 2009).

In the AD, VFAs are an essential intermediate product. VFA occurs in digesters as free VFA and ionized VFA, the equilibrium of which is pH dependent (Kunte et al. 2000b). The followings are a summary of the effects of VFA inhibition: (1) influence on cell electrophysiology and metabolism; (2) cytoplasm acidification; and (3) cause osmotic problems.

Because free VFA is lipophilic and can readily penetrate the cell membrane, it is regarded as more harmful to pathogens than ionized VFA, whereas ionized VFA is lipophobic and cannot pass across the cell membrane (Puchajda et al. 2006). Most bacteria have a negative charge and an internal pH that is neutral or slightly alkaline, which can create a particular proton driving force (PMF, p) across the cell membrane. Free VFAs pass through cell membranes and dissociate into the more alkaline inner section to create H+ and ionized VFAs, which acidify the cytoplasm at low ambient pH (Magnowska et al. 2019).

The PMF, consisting of a pH gradient (ΔpH) and electrical potential (Δψ) throughout the cell membrane, plays an important role in the metabolism of bacteria (Alwazeer & Cachon 2020). For instance, it is reported that PMF affects the sensory transduction of bacteria, stimulates the synthesis of adenosine triphosphate (ATP), activates the active transport of ions and metabolites, provides mechanical force for flagellate engines, controls the expression of Staphylococcus aureus lrgAB, and controls DNA absorption during genetic transformation (Magnowska et al. 2019; Yavitt et al. 2020). Reducing PMF can cause great harm to the metabolism of pathogens, but the detailed mechanisms are still unclear.

Mills et al. (2004) suggested that VFA can change the structure of the cell membrane or unlock the electronic transport system to prevent the generation of ATP and the transport of the necessary compounds. They also reported that VFA strongly inhibits the uptake of L-serine or other L-amino acids in Bacillus subtilis. Dissociated H+ in cells can acidify cytoplasm. To overcome this problem, cells must pump H+ through a mechanism called an H + -ATP pump, which consumes energy and can lead to energy depletion of the cell (Puchajda & Oleszkiewicz 2006).

The inhibitory impact of VFA on pathogen inactivation is attributable to all three factors and varies depending on parameters such as pathogen kind, VFA type, VFA concentration, and pH level. Salmond et al. (1984) studied the impact of low benzoate concentration (2.5 mM) on E. coli in a pH range of 5.0–7.6 and revealed that while benzoic acid had a considerable inhibitory effect on cytoplasmic acidification, its effect on cell metabolism was greater. Eklund et al. (2005) investigated the suppression of sorbic acid by various bacteria and discovered that both free and ionized acids were involved. Although the inhibitory impact of free acid is 10–600 times larger than that of ionized acid, ionized acid inhibits growth by 50% at pH > 6.

Sheu and Freese (1972) found that the inhibition of 0.2 M acetate on Bacillus subtilis was reduced but not eliminated by adding 0.2% glucose or fructose. He also noted that when cells were transferred on without VFA after exposure to 200 mM acetate for 2 h, the inhibited cells grew at the same rate as untreated cells. But Bai et al. (2021) reported that VFA could acidify E. coli's cytoplasm and cause a decrease in glutamate in the cell when cells were transferred from VFA to a medium without VFA, the recovery of cytoplasmic pH depended on glutamate synthesis. Boon et al. (2020) reported that the ATR of Salmonella typhimurium was caused by extreme acid shock with pH 4.4, but not in sub-inhibitory VFA concentrations.

This review paper has demonstrated that VFA can be used to inactivate pathogens present in FS, besides its applications, VFA can be also produced from different wastes through acidogenic fermentation and it can be also used as a carbon substrate for the production of bio-plastics, bioenergy, and the biological removal of phosphorus and nitrogen. To increase the production of VFA various methods can be used, including pre-treatment methods and chemical methanogenic inhibitors. High concentrations of VFAs and ammonia are important factors contributing to pathogen inactivation during AD, particularly low pH VFAs, and high pH ammonia. VFA treatment with elevated temperature can significantly reduce pathogens. However, the degree of reduction can be varied depending on the concentration of organic acid, pH, and temperature. A concentration of 4,800–6,000 mg/L, was required to achieve 10 E. coli log inactivation and complete A. lumbricoides egg inactivation in 4 days. To achieve E. coli log inactivation in the range of 3–5 in 4 days, a concentration of around 2,800–4,300 mg/L was required.

The authors would like to acknowledge the project support of the National Key Research and Development Plan (2019YFC0408700), the Fundamental Research Funds for the Central Universities (FRF-DF-19-001).

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.