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Article

Use of Alcaligenes faecalis to Reduce Coliforms and Enhance the Stabilization of Faecal Sludge

by
Nataliya Loiko
1,*,
Oleg Kanunnikov
2 and
Yuriy Litti
1,*
1
Winogradsky Institute of Microbiology, Federal Research Center «Fundamentals of Biotechnology», Russian Academy of Sciences, Moscow 117312, Russia
2
Rail Chemical LLC, Moscow 105005, Russia
*
Authors to whom correspondence should be addressed.
Sustainability 2023, 15(16), 12580; https://doi.org/10.3390/su151612580
Submission received: 17 July 2023 / Revised: 12 August 2023 / Accepted: 16 August 2023 / Published: 18 August 2023
(This article belongs to the Section Resources and Sustainable Utilization)
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Abstract

:
The disposal of human faecal sludge (FS) is one of the biggest environmental problems. It can be solved by using FS as an agricultural fertilizer. However, this is hampered by the presence of pathogenic microflora and high organic matter content in FS. This paper proposes a novel treatment approach for FS to address these issues. It consists of the preliminary aerobic incubation of FS with the bioaugmentation of bacterial culture Alcaligenes faecalis DOS7. A. faecalis has been shown to inhibit the growth of various microorganisms, including coliforms (Escherichia coli). For the treatment of FS, three tanks with a volume of 1 m3 each, equipped with a mixing and aeration system, were used. A. faecalis culture was introduced into two experimental tanks at a concentration of 106 and 6.5 × 106 cells/mL. The 11-day incubation in the experimental tanks resulted in the decomposition of organic matter in the FS that was several times faster than in the control (p < 0.05). Total suspended solids decreased 2.5–5-fold, chemical oxygen demand decreased 1.8-fold, 5-day biochemical oxygen demand decreased 1.5–2-fold. At the same time, after 4 days of incubation, no coliforms were detected in the experimental tanks, and in the control, coliforms accounted for 13.9% of the total number of cells after 11 days of incubation. The proposed method of FS pretreatment is a real alternative to the existing ones and can be used both individually and in combination with other methods, for example, composting.

Graphical Abstract

1. Introduction

The recycling and disposal of faecal sludge (FS) is a major problem of human society [1,2,3]. When accumulated in cesspools or lagoons, it is a source of stench and infections [2]. At the same time, most FS eventually ends up in rivers and seas, where it poses a significant environmental threat. However, FS can be effectively utilized to produce biogas and biochar, as well as construction materials [4,5,6,7,8]. In addition, FS can be successfully used in agriculture as a soil conditioner/fertilizer, which is especially relevant for agricultural countries [9]. FS contains many useful substances: proteins, fatty acids, undigested cellulose residues, pigments, vitamins, etc. [10,11]. In addition, it is rich in nitrogen (N), phosphorus (P) and potassium (K), which are necessary biogenic elements for increasing soil fertility [12].
However, the direct use of human faeces as fertilizer is extremely undesirable. First, the organic composition of FS is not stabilized, and applying it to the soil without special treatment can be harmful to crops [12]. Second, FS is characterized by an abundance of pathogenic microorganisms, such as Salmonella, Escherichia coli, and faecal streptococci and staphylococci, and it can also be contaminated with helminth eggs [13]. When faecal matter is introduced into the soil, the pathogens present in it contaminate the soils and infect the people who cultivate the land [14,15]. Therefore, the safe use of FS in agriculture is possible only after special treatment procedures, leading to a reduction in the number of pathogenic microflorae [16].
There are various chemical and biological methods for reducing the infectious potential of faeces, which have their pros and cons [17,18]. For example, the feasibility of treating FS with ammonia has been reported. It should be noted that if the faeces are not too diluted, the urine contained in it (at an ammonia concentration of more than 40 mM) is sufficient to inactivate the bacterial pathogens. However, higher concentrations of ammonia, elevated temperatures and the long-term storage of the waste for 1–6 months are needed to kill any ascarid eggs [19]. The use of synthetic urea complicates FS treatment technology, reduces economic feasibility and increases ammonia odours [20]. Another way to reduce the infectious potential of FS is the addition of lime [21]. Within eight days of stabilization in alkaline conditions, E. coli, faecal Streptococcus, faecal coliforms and Salmonella are entirely devitalized [22]. However, this method has its disadvantages. A constant addition of lime is required to maintain the pH of the FS at an alkaline level, and its constant application leads to the formation of limescale [20]. In this regard, in practice, it is proposed that combined methods should be used for converting FS into fertilizer, for example, composting with additional leaching with lime [23]. Such methods also have their disadvantages. They require additional equipment costs, are long-term, and also do not sufficiently reduce the infectious component of the faecal matter.
At present, the focus of attention regarding the problem of processing FS to obtain high-quality agricultural fertilizers is shifting towards the use of more sustainable microbiological approaches. Lactic acid fermentation has been shown to be promising for reducing the infectious potential of FS [9,24,25,26]. For example, Odey et al. showed that the addition of lactic acid bacteria to the FS initiated lactic acid fermentation, leading to a pH reduction to the value of four [24]. After 7 days of the process, the number of faecal coliforms decreased by half, and after 15–17 days, this value fell below the detection limit. The authors concluded that faecal coliforms were destroyed due to acidification. However, a number of studies have noted that many lactic acid bacteria are not able to acidify FS sufficiently to significantly reduce the number of pathogens, so it is suggested that the lactic acid or substrates (fermented rice flour or cassava flour) that activate the growth of lactic acid bacteria should also be added [26]. Using combined methods is also recommended, such as lactic fermentation with the composting processes [27]. All these measures increase the cost and time of FS processing. In addition, it has been shown that the direct use of lactic acid fermented faeces in agriculture may be limited due to incomplete decomposition, high concentrations of organic acids or insufficient hygienic treatment [9].
Thus, the search for low-cost and effective alternatives to the treatment of FS is still an important issue in order to prevent risks to human health and ensure its safe use as a soil fertilizer. As an alternative to the use of lactic acid (or other pH-lowering acid, e.g., propionic acid [28]), the fermentation of FS could be carried out through the use of biological preparations based on non-pathogenic hydrolytic bacteria, which could not only reduce the infectious potential of FS, but also lead to the partial degradation of polymeric organic matter in FS.
Previously, bacterial strain Alcaligenes faecalis DOS7 was isolated and identified. A. faecalis DOS7 is non-pathogenic for warm-blooded animals and is resistant to the actions of quaternary ammonium compounds (QAC) and polyhexamethylene guanidine (PHMG) [29]. It was discovered that A. faecalis is also quite resistant to the action of various stresses, such as exposure to pH or thermal shock, antibiotics or oxidizing agents and high salt concentrations [30]. Under laboratory conditions, it has been shown that A. faecalis can grow successfully on the organic matter of FS, especially under aerobic conditions [29]. Also, as a chemoorganoheterotroph, A. faecalis is capable of utilizing various substrates: acetate, propionate, butyrate, citrate, alanine and certain other amino acids. A. faecalis can also decompose urea, which is present in large quantities in FS, to produce ammonia, which raises pH. At the same time, A. faecalis is resistant to alkaline pH, maintaining a neutral pH in its cytosol to prevent the damage and denaturation of macromolecules. These characteristics of the isolated strain make it promising for use as a bacterial preparation for enhancement of FS treatment in order to obtain high-quality and safe fertilizers.
Therefore, the goals of this research were (1) to study the antagonistic properties of A. faecalis against various groups of microorganisms, including coliform bacteria; (2) to evaluate the feasibility of using A. faecalis for improving the efficiency of FS treatment and reducing its infectious potential.

2. Materials and Methods

2.1. Bacteria

The objects of the study were Gram-negative bacteria Alcaligenes faecalis DOS7, isolated from FS [29,30].
For experiments to determine microbial antagonism, test microorganisms were provided by the collection of the Research Center for Biotechnology, Russian Academy of Sciences:
  • Gram-negative bacteria Escherichia coli K12 MG1655 and Pseudomonas aeruginosa 4.8.1;
  • Gram-positive non-sporulating bacteria Micrococcus luteus NCIMB 13267 and Staphylococcus aureus 209P;
  • Yeast (eukaryote) Yarrowia lipolytica 367-2.

2.2. Cultivation

The collection and isolated bacterial strains were grown in a lysogenic broth (LB) medium (Broth, Miller, VWR Life Science, Radnor, PA, USA). Bacteria were cultivated in 250 mL flasks containing 50 mL of nutrient medium at 28 °C and stirred on an orbital shaker (120 rpm) for 1 day (until the stationary phase). A total of 0.25 mL of the culture of the beginning of the stationary phase of growth (inoculum) was added to 50 mL of the medium (0.5% vol.).

2.3. Faecal Sludge

FS samples were taken at the beginning of January 2022 from the trains of the North-Western branch of Federal Passenger Company JSC (Moscow, Russia). Specifically for this study, FS was not treated with any biocides.

2.4. Determination of Microbial Antagonism

2.4.1. Perpendicular Strokes Method

The studied antagonist bacteria A. faecalis was streaked onto the surface of the agar medium in a Petri dish. Seeding was performed on the diameter of the Petri dish which was then placed in the thermostat at 28 °C for 1 day. During this time, A. faecalis bacteria grew in the form of a long streak in the middle of the Petri dish. Then, perpendicular to the grown streak, test microorganisms were seeded with strokes, starting from the edges of the Petri dish. Petri dishes were placed in the thermostat for 48 h. Then, the distance (in mm) at which the growth of test microorganisms started from the growth of A. faecalis was measured. The greater this distance, the more sensitive the test microorganism to the substances produced by A. faecalis [31].

2.4.2. Disk Diffusion Test

A. faecalis bacteria were grown for 1 day as described above. Then, the culture fluid was centrifuged at 12,000× g for 10 min to separate the cells. The supernatant was additionally filtered on 200 nm filters (EMD Millipore Corporation, Bedford, MA, USA). Test organisms were grown to a stationary growth phase as described above. Then, 0.1 mL of microbial suspension (about 108 cells) was transferred to a Petri dish containing LB agar medium and evenly distributed over the entire surface of the dish. Then, sterile paper discs (concentration disks 1/4″, Difco Laboratories, Detroit, MI, USA) were placed on the agar, on which 10 µL of the prepared A. faecalis culture fluid was applied. The growth retardation zone around the disks was determined after the plates were incubated in a thermostat at 28 °C for 48 h. The zone diameter (in mm) was used to assess the bactericidal effect of FS [29].

2.5. Conducting an Experiment on the Biodegradation of FS Using A. faecalis

For the experiment, three folding tanks made of a 1 m3 polyvinyl chloride (PVC) membrane were specially designed and constructed. The tanks were installed in a warm room and operated at a temperature of 20 °C. The tanks were equipped with a recirculation system using a drain pump and an aeration system employing an aquarium compressor with a capacity of 600 L/min. The mixing mode was regulated by a timer according to the following program: 30 min mixing, 30 min idle (to cool the pumping equipment). The general view of the experimental tanks is shown in Figure 1.
Each tank was loaded with 0.8 m3 of FS. Then, 0.4 and 2.6 L of A. faecalis bacterial preparation with a cell titer of (2.0 ± 0.3) × 109 cells/mL, grown as described earlier, were added into the second (Tank2) and third (Tank3) tanks, respectively,. The first tank (Tank1), without the addition of the bacterial preparation, served as the control tank. After the addition of the bacterial preparation, the A. faecalis cell titer in Tank2 was ~106 cells/mL, and in Tank3 it was ~6.5 × 106 cells/mL. Next, a mixing and aeration system was run in all three tanks.
Incubation was performed for 11 days. FS samples were taken for microbiological and chemical analysis on days 1, 3, 6, and 11. A zero sample was taken 40 min after the beginning of the experiment.

2.6. Determination of Colony-Forming Units

Samples of FS after appropriate dilution were plated on LB agar nutrient medium in Petri dishes. Petri dishes were incubated for 3 days at 28 °C. The number of colony-forming units (CFU) in the dilutions was counted, and after, that the number of cells (in CFU/mL FS) was determined.

2.7. Tests for Coliform/E. coli

Coliforms (including E. coli) decompose lactose on m-Endo agar, resulting in the formation of acetaldehyde along with various aldehydes. This results in the formation of red colonies with a green sheen. Non-coliform organisms form colourless colonies on m-Endo-agar; this is the basis for their differentiation from coliforms, including E. coli [32].
Aliquots of FS were plated after appropriate dilution on ENDO agar in Petri dishes (Mersk KGaA, Darmstadt, Germany). The incubation of Petri dishes was carried out at 28 °C for 4–6 days. The total number of colonies and the number of colonies with green sheen corresponding to coliforms were then counted. The percentage of coliforms relative to the total number of colonies grown was calculated.

2.8. Determination of Bactericidal (MBC) and Minimum Inhibitory (MIC) Concentrations

Solutions of biocides/antibiotics were added at various final concentrations to 25 mL glass tubes containing 5 mL of LB medium. The tubes were inoculated with A. faecalis in a stationary growth phase, closed with cotton plugs and incubated with 120 rpm agitation at 28 °C. The growth of A. faecalis was assessed visually by the appearance of turbidity after 1 day of incubation. The lowest concentration of biocide at which no bacterial growth was observed was taken as the MIC. Also, after 2 days of incubation, aliquots were inoculated from test tubes in which the visual growth of bacteria was not observed on LA agar nutrient medium. The lowest concentration of the biocide in the test tube, at which no growth of A. faecalis was observed on the agar medium, was taken as MBC.

2.9. Analytical Methods

The FE20 pH meter (Mettler Toledo, Greifensee, Switzerland) was used for pH measurement. Ammonium and bicarbonate were determined using capillary electrophoresis system Capel-205 (Lumex, St. Petersburg, Russia). Untreated fused silica capillaries (60 cm length, 75 μm internal diameter) were used for electrophoresis. The capillary was held at 20 °C and the applied voltage was 13 kV and −17 kV for ammonium and bicarbonate, respectively. Chemical oxygen demand (COD), total suspended solids (TSS) and total nitrogen (Ntot) were determined using standard methods [33]. The WTW OxiTop respirometric BOD measuring system (Weilheim, Germany) was used for the determination of five-day biochemical oxygen demand (BOD5).

2.10. Statistical Methods

Differences in biochemical parameters and cell titre among tanks and antagonistic characteristics of A. faecalis among test cultures were tested with a one-way analysis of variance (ANOVA). Tukey’s post hoc tests were conducted when the differences were statistically significant at a level of p < 0.05. All statistical analyses were performed using Microsoft Excel 2019 software.

3. Results

3.1. Antagonistic Properties and Resistance of A. faecalis DOS7

3.1.1. Perpendicular Strokes Method

For a rapid screening of the antagonistic properties of A. faecalis, the perpendicular strokes method was used [31]. Test microorganisms were seeded perpendicularly to the grown A. faecalis bacteria after their stroke seeding. The antagonistic abilities of the test microorganism were judged by the distance over which the growth of the test microorganism was inhibited (Figure 2). The test microorganisms chosen were opportunistic bacterial strains frequently encountered in FS whose pathogenic counterparts are the causative agents of human and animal infectious diseases: Gram-negative P. aeruginosa and E. coli and Gram-positive S. aureus. In addition, it is by the number of coliform bacteria, which include E. coli bacteria, that water contamination was judged. The test microorganisms also included the bacteria M. luteus, which is a human symbiont and is present in large numbers in the FS, as well as yeast, as representatives of eukaryotes.
A. faecalis was found to exhibit antimicrobial properties against all microorganisms used in the test, but the greatest effect was obtained against the Gram-positive bacteria S. aureus and M. luteus (Table 1). The distance by which their growth was inhibited in the perpendicular stroke method was 18 and 26 mm, respectively. With respect to Gram-negative bacteria E. coli and P. aeruginosa and yeast, the antagonistic ability of the studied strain was less prominent, but was also reliably detected.

3.1.2. Disk Diffusion Test

The above result was confirmed in another experiment using the disc diffusion method. The object of the study was culture fluid after 1 day of A. faecalis growth, completely freed of cells by centrifugation and filtration. It was applied (10 µL each) to cotton disks placed on a freshly seeded lawn of test microorganisms. The antagonistic property of A. faecalis culture fluid was judged by the area of growth inhibition around the disc. Again, the effect was more pronounced against Gram-positive bacteria (Figure 3, Table 1). In the case of P. aeruginosa, the zone of partial growth suppression was smaller.

3.1.3. Resistance of A. faecalis Bacteria to the Action of test Microorganisms, Antibiotics and Biocides

It is crucial to note that the “reverse” experiment demonstrated that the A. faecalis bacteria were not sensitive to the test microorganisms. A. faecalis was co-dispersed on the agar medium with P. aeruginosa, E. coli, and S. aureus so that its growth was delayed (seeding from suspension with glycerol, stored for 1 month at −20 °C) after the appearance of bacterial colonies of test microorganisms. The growth resumed after the appearance of A. faecalis colonies, without the zones of growth suppression but rather with “crawling” on the colonies of test microorganisms (Figure 2, lower right image). As a result, it was demonstrated that A. faecalis DOS7 exhibits antagonistic characteristics against a variety of microorganisms, including coliforms, while being insensitive to the growth of these microorganisms.
Additionally, A. faecalis DOS7 displayed high MIC and MBC values against a variety of biocides and antibiotics, suggesting a significant resistance to their action (Table 2). This is important because FS often contains antibiotics and biocides that can inhibit the activity of biological preparations.

3.2. Efficiency of FS Degradation with A. faecalis

The usefulness of A. faecalis as a biological preparation for the treatment of FS was examined after proving the antagonistic capabilities of the organism against several test organisms and exhibiting resistance to their effects. Three 1 m3 tanks (Tanks) equipped with circulation and aeration systems were assembled into a pilot plant for this purpose (Figure 1). Each of the tanks received 0.8 m3 of fresh FS, and Tank2 and Tank3 also received 0.4 and 2.6 L of the A. faecalis bacterial preparation (2 × 109 cells/mL), respectively. Tank1 was the control tank, without the addition of A. faecalis. It is feasible to assess the efficacy of FS bioconversion by observing the change in the major biochemical parameters during the course of the incubation, which lasted 11 days and involved constant stirring and aeration. Thus, the concentrations of TSS, COD, and BOD5 were determined. The more active the microbial processes in FS, the more suspended solids are converted to dissolved organic matter (solubilization process). Also, due to the consumption of organic matter by microorganisms, COD (reflects the total content of oxidizable organic matter, including difficult (e.g., polyaromatic or humified compounds and easily degradable (sugars, acids, alcohols, etc.) compounds) and BOD5 (reflects the content of organic and some inorganic substances that are metabolized by microorganisms while consuming a certain amount of oxygen) become reduced. The progress of the decomposition of urea, which is present in significant amounts in the FS, can be determined by monitoring changes in ammonium, Ntot, and bicarbonate during incubation [34].

3.2.1. Dynamics of the Main Biochemical Parameters of FS Biodegradation

TSS decreased by 10% on average after the first day of incubation, with a slightly larger reduction in the control tank (Table 3). After 4 days, however, TSS in Tank2 decreased by 47% of the initial value, and in Tank3 by 55%. After 11 days, TSS already decreased by 65% and 81%, respectively. A substantially lesser decrease in TSS was observed in the control Tank1 at the end of 4 and 11 days: 21 and 30%, respectively (p  <  0.05).
These findings demonstrated the excellent efficacy of FS pretreatment with bacterial preparation containing A. faecalis. Moreover, the effect was dependent on but not directly proportional to the initial number of added bacteria. The number of A. faecalis bacteria added to Tank2 was 6.5 times lesser than that added to Tank3, and the difference in the reduction in suspended solids between Tank2 and Tank3 was only 1.25-fold by the 11th day (p < 0.05). This indicates that in the future, it will be possible to optimize the dosage of the biological preparation. Visually, the processes taking place were recorded through the clarification of the FS samples taken from the tanks for analysis. The samples from the experimental tanks started off with the same dark brown colour, but after 4 days (and up until the finish), they all changed to a substantially lighter shade (Figure 4). In addition, their consistency became less saturated.
The COD results verified that the FS biodegradation in the experimental tanks improved. The progressive decline in COD levels throughout the course of incubation is thought to be the result of aerobic bioconversion of organic matter to CO2, which then escaped from the open tanks into the atmosphere. The COD drop in the control Tank1 was 16% in 11 days, which was substantially different from the COD removal in Tank2 and Tank3 (p  <  0.05), while in the experimental Tank2 and Tank3, COD reduction was 44 and 45%, respectively. Therefore, for this indicator, there was no discernible difference between the experimental tanks.
Up to day 6 of incubation, no significant changes in another indicator, BOD5, were observed in any of the tanks (Table 3). However, on day 11, it considerably dropped by 1.5 and 2 times in the experimental Tanks 2 and 3, respectively; the BOD5 in Tank3 was 1.67 times lower than in the control Tank1 (p < 0.05). This suggested that there was less organic material available for microbial conversion. In the experimental Tank1, a decrease in BOD5 was only recorded by a factor of 1.2 within 11 days.

3.2.2. Dynamics of Biochemical Indicators Associated with the Bioconversion of Nitrogen and Bicarbonates

In order to better understand the processes of FS biodegradation, the dynamics of biochemical parameters related to nitrogen and bicarbonate metabolism were additionally tracked (Table 4). All test samples had a tendency to have lower total nitrogen concentrations, demonstrating that microbial bioconversion uses nitrogen. As was previously demonstrated, microbial activity was more prominent in the experimental samples, where the reduction was more dramatic. Around 90% of the total nitrogen was initially in the form of ammonia. It ranged between 97 and 99% in the experimental samples after one day, while it stayed at 92% in the control tank (Tank1). The biodegradation of urea, which is present in significant amounts in FS [34], is the cause of this tendency.
Throughout the entire experiment, the amount of bicarbonate also reduced in all three tanks, seemingly as a result of its consumption by microbial biomass and/or stripping. This reduction was not significant in the control tank at 25%, but it was 39 and 45% in the experimental tanks. After the sixth day of incubation, the bicarbonate concentration in Tank3 was significantly, 1.4–1.5 times lower compared to that of the control Tank1 (p  <  0.05). Once more, it was obvious that the A. faecalis addition was superior to the control. Furthermore, all test tank pH levels remained stable during the experiment (Table 4), which is important to highlight.
Thus, based on the changes in the biochemical characteristics determined in this work, it was shown that more active microbial biodegradation processes take place in the experimental tanks. At the same time, there is no discernible difference in the biodegradation activity between Tank2, which had less A. faecalis bacterial preparation added, and Tank3, which received a greater dose.

3.3. Changes in the Total Cell Number and Infectious Potential of FS

By periodically seeding the chosen aliquots onto nutrient-dense medium (CFU method), the microbial titre was assessed in order to understand the changes in the microflora in the control and experimental tanks during incubation. Simultaneously, the number of coliform bacteria was determined in these aliquots (by seeding on Endo agar), which provided an indication of the change in the infectious potential of the FS during the experiment. Coliform bacteria are prevalent in the FS and reside in the gastrointestinal system. They are employed as proof of faecal contamination in environmental samples because it is time- and money-consuming to detect all pathogen species [21].
Initially, 40 min after biological preparation application to Tank2 and Tank3, their microbial titre was higher than that of the control, ~1.5 and 2.7 times greater (p  <  0.05), respectively, and coincided with the expected calculated values (Table 5). The microbiota in all three samples may be described as being highly active and diverse. The screenings of Samples 2 and 3 revealed colonies of A. faecalis. On the endo agar, up to 30% (of the total number) of coliform bacteria with a distinctive metallic or greenish sheen were seen (Figure 5).
The introduction of new bacterial cells and intense aeration, which led to some cells failing to form a colony on the agar media, are likely to be responsible for the microbial titer in the experimental samples decreasing one day after the experiment began. However, in all three tanks, the number of microorganisms was detected at 4.0 × 106 cells/mL. The number of coliforms (as a percentage) decreased insignificantly in the control, whereas in the experimental samples, it decreased almost fourfold.
The number of cells in the control samples remained constant after 4 days, whereas it doubled in the experimental samples (p  <  0.05). The coliform bacteria in the control tank were found to be 20% of the total number of cells, while in the experimental tank they were no longer detected. By day 11, the microbial titer in Tank 1 continued to decrease, while in experimental Tanks 2 and 3 it remained at a high level. Only the control still had coliforms present. The spreading colonies of A. faecalis were easily seen when plating on LB agar media (Figure 5, bottom right image).
As a result, the experiment’s outcome was quite positive. Microbial activity in the experimental tanks remained at a high level throughout the experiment, with coliforms being undetectable as early as 4 days after the start of the experiment.

4. Discussion

Finding rapid, efficient, and environmentally appropriate ways to transform FS into secure agricultural fertilizer was the major goal of this research. The only technique suggested up to this point was the employment of lactic acid bacteria to acidify FS to pH 4 [24,25,26], which is an alternative to composting (a prolonged process) [23] and chemicals (which are not environmentally friendly). The effectiveness of this treatment in reducing the infectious potential of FS, where harmful faecal bacteria do not multiply or are eliminated, is its main strength (the Introduction presents this information in greater depth). The organic matter in the FS is not stabilized by this method, however. Additionally, the soil can suffer after application of an acidic pH.
It was decided to investigate the feasibility of employing the A. faecalis strain DOS7, previously isolated from FS, to accelerate the biodegradation of FS because it demonstrated good productivity while actively growing under aerobic circumstances in the faecal environment. In biotechnology, different strains of A. faecalis are frequently utilized, particularly for wastewater treatment and soil bioremediation. For instance, it has been suggested that the A. faecalis strain HP8 can biodegrade anthracene in soil [35], A. faecalis JQ191 may do the same for quinolinic acid [36], and A. faecalis RB-10 can do the same for indole [37]. A. faecalis strains like HO-1 have the ability to directly oxidize ammonium to nitrogen gas (Dirammox) [38]. Simultaneous ammonia nitrogen and organic carbon removal in a biofilm reactor was achieved after bioaugmentation with A. faecalis strain NR [39]. At the same time, there are strains that can cause human infectious diseases [40]. According to toxicological testing, the strain utilized in this study is not harmful to people or animals [29]. Additionally, it demonstrated extremely strong resistance to biocides like QAC and PHMG, which is also a benefit because FS can come into contact with any antibiotic or biocidal substance.
The initial goal of this work was to demonstrate the antagonistic qualities and ability to suppress the growth of pathogens of the A. faecalis DOS7 bacteria suggested for the novel approach. This task was perfectly performed by A. faecalis DOS7. It exhibited antimicrobial properties against all the microorganisms used in the test, among which were opportunistic bacteria. Its antagonistic properties were most strongly demonstrated against Gram-positive bacteria. The insensitivity of the A. faecalis cells to other organisms was also crucial. When spread out on Petri dishes with other bacteria, they did not exhibit sensitivity; rather, they started to take over the entire area and, in addition, to develop over other colonies, crawling over them. Additional experiments showed that the strain under study was resistant not only to QAC and PHMG, but also to a number of other biocidal substances and antibiotics. All these properties exhibited by A. faecalis DOS7 make it promising for use in the management of FS.
Incubation was carried out at the pilot level in three tanks with a volume of 1 m3 with mixing and aeration. Therefore, approximately the same results can be obtained at the industrial level. Aeration not only promoted the growth of the added A. faecalis, but also fulfilled an additional function of reducing the infection potential, as pathogenic bacteria do not like aeration. The ability of A. faecalis to grow under microaerophilic conditions has already been demonstrated [29]; therefore, biodegradation processes would still occur even if aeration and agitation could not be managed.
Coliforms were used as indicator organisms to assess the reduction in infection potential, which is a common practice [20]. In the experimental tanks, they disappeared already 4 days after the start-up. In the control tank, they were detected after 11 days in the amount of 14% of the titer of all microorganisms (at their initial content of 30.5%). Apparently, their decrease in control was due to a sufficiently strong aeration of the FS, as discussed above. In addition to reducing the infectious potential, A. faecalis significantly removed the solid content of FS, reducing TSS by 2.5–5 times. The heterogeneity of the FS in the experimental tanks decreased so that their colour became much lighter. This is also advantageous since a more uniform suspension is more evenly disseminated among soil particles when used as fertilizer. The amount of organic matter in the experimental samples after incubation was greatly reduced, as evidenced by the determination of COD and BOD5 indicators, which lessens the risk of “over-fertilizing” the soil and “burning” plants. As they are regarded as rhizobacteria that promote plant growth, A. faecalis bacteria with treated FS can also have a positive impact on plants [41]. These bacteria can produce phytohormones, ACC deaminase, and indole acetic acid, which alter plant metabolism and morphology and increase yields and growth metrics [42].
In general, the obtained data provide a good basis for the creation of a new technology for the processing of FS by incubation not only with A. faecalis, but also with other promising strains of soil bacteria. The suggested approach does not necessitate high start-up or ongoing operating expenditures. The A. faecalis DOS7 strain is quite easy to cultivate and store. It can also work in low-oxygen environments, although not as efficiently. Therefore, to improve the qualities of FS, it is conceivable to introduce these bacteria in the form of a biopreparation directly into cesspools or lagoons if it is not possible to equip special aerated tanks, as was performed in our work. It is not possible to assess the overall cost, time, and effectiveness of FS pre-treatment by different approaches based on the body of published literature. The proposed technology’s optimization and the application of the pretreated FS as fertilizers will be the main areas of future investigation.

5. Conclusions

There are numerous approaches in use today for managing human FS, the amount of which is constantly rising and posing harm to the environment. Processing FS into fertilizers for use in agriculture is a sustainable solution for its disposal. This requires stabilizing organic matter, increasing the availability of organic matter for plants, and lowering the high infectious potential of FS (i.e., reducing pathogens). The use of aerobic incubation with bioaugmentation of the A. faecalis DOS7 culture, which encourages effective biodegradation of organic matter and suppression of pathogenic coliforms, is a novel method for the exploitation of FS proposed in this work. It has been demonstrated that A. faecalis is resistant to the effects of several biocides and antagonistic to a variety of test bacteria, including coliforms. In a 1 m3 tank with a mixing and aeration system, the aerobic treatment of FS with the addition of A. faecalis produced statistically substantially (p < 0.05) superior results in the removal of organic matter and the reduction in coliforms when compared to the control. The suggested method worked well even at low concentrations of A. faecalis (up to 106 cells/mL). The suggested method has a number of benefits, including a decrease in the infectious potential, stabilization of organic matter for safer plant use, a decrease in heterogeneity that increases the efficacy of FS when applied to soil. An additional introduction of A. faecalis into the soil may promote plant growth and stress resistance. The suggested approach is also simple to implement, which lowers the financial costs of application. This FS pretreatment approach can also be used in conjunction with alternative methods such as composting. In this case, A. faecalis bacterial preparation can be added directly to the compost piles to enhance the biodegradation of FS.

Author Contributions

Conceptualization, N.L. and Y.L.; methodology, N.L. and Y.L.; formal analysis, O.K; investigation, N.L. and Y.L.; resources, O.K.; writing—original draft preparation, N.L.; writing—review and editing, N.L. and Y.L.; visualization, N.L.; supervision, O.K.; project administration, O.K.; funding acquisition, O.K. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Ministry of Science and Higher Education of the Russian Federation. Chemical analyses were partially supported by grant no. 075-15-2022-318, dated 20 April 2022, through state support for the creation and development of a world-class scientific centre “Agrotechnologies for the Future”.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Acknowledgments

We sincerely thank Dmitry Serdyukov for his help in experiments.

Conflicts of Interest

The authors declare that they have no known competing financial interest or personal relationships that could have appeared to influence the work reported in this paper.

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Figure 1. General view of the experimental setup of three folding PVC membrane tanks with a volume of 1 m3.
Figure 1. General view of the experimental setup of three folding PVC membrane tanks with a volume of 1 m3.
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Figure 2. Screening of the antagonistic properties of A. faecalis against test microorganisms by perpendicular strokes method. The names of the test microorganisms are signed in the subfigures. The lower right figure is an illustration of the insensitivity of A. faecalis to the action of test microorganisms.
Figure 2. Screening of the antagonistic properties of A. faecalis against test microorganisms by perpendicular strokes method. The names of the test microorganisms are signed in the subfigures. The lower right figure is an illustration of the insensitivity of A. faecalis to the action of test microorganisms.
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Figure 3. Zones of growth suppression of test microorganisms by the A. faecalis culture fluid. The names of the test microorganisms are signed at the top.
Figure 3. Zones of growth suppression of test microorganisms by the A. faecalis culture fluid. The names of the test microorganisms are signed at the top.
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Figure 4. Colour change in FS during incubation (from left to right: Tank1, Tank2 and Tank3).
Figure 4. Colour change in FS during incubation (from left to right: Tank1, Tank2 and Tank3).
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Figure 5. View of colonies on Petri dishes with LB (light) and ENDO agar (red).
Figure 5. View of colonies on Petri dishes with LB (light) and ENDO agar (red).
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Table 1. Antagonistic characteristics of A. faecalis. Means ± standard deviations (SD) are given. Different letters reveal a significant difference from post hoc Tukey’s test analysis.
Table 1. Antagonistic characteristics of A. faecalis. Means ± standard deviations (SD) are given. Different letters reveal a significant difference from post hoc Tukey’s test analysis.
Test CultureDistance by Which the Growth of the Test Culture is Inhibited in the
Perpendicular Strokes Experiment, mm		Diameter of Growth Inhibition Zone in the Disc Diffusion
Experiment, mm
P. aeruginosa7 ± 1 ac15 ± 3 a,*
E. coli11 ± 2 abc11 ± 2 a
S. aureus18 ± 2 abc19 ± 2 a
M. luteus26 ± 3 b25 ± 3 a
Y. lipolitica5 ± 1 ac11 ± 1 a
* Diameter of the partial growth suppression zone.
Table 2. MIC and MBC of antibiotics/biocides in relation to A. faecalis DOS7.
Table 2. MIC and MBC of antibiotics/biocides in relation to A. faecalis DOS7.
No.Antibiotic/BiocideMIC, ppmMBC, ppm
1Ciprofloxacin16.3 19.6
2Tetracycline30.0 35.0
3Sodium dehydroacetate 45,000>100,000
4Bronopol4040
52,2-dibromo-3-nitrilopropionamide2525
6Sharomix600600
7Sodium percarbonate50006000
8Silver citrate15,00025,000
9Didecyldimethylammonium chloride900900
10Alkyldimethylbenzylammonium chloride 600600
11Polyhexamethylene guanidine500500
12Chlorhexidine digluconate10002000
Table 3. Dynamics of the main biochemical parameters of the FS incubation process in control and experimental (with the addition of A. faecalis) tanks. Means ± standard deviations (SD) are given. Different letters reveal a significant difference from post hoc Tukey’s test analysis.
Table 3. Dynamics of the main biochemical parameters of the FS incubation process in control and experimental (with the addition of A. faecalis) tanks. Means ± standard deviations (SD) are given. Different letters reveal a significant difference from post hoc Tukey’s test analysis.
Incubation Time, DayTSS, mg/L COD, mg/LBOD5, mg/L
Tank1Tank2Tank3Tank1Tank2Tank3Tank1Tank2Tank3
05362 ± 2455641 ± 2295477 ± 2868600 ± 3788712 ± 3928464 ± 3384050 ± 2064242 ± 2354117 ± 199
14715 ± 1925038 ± 2105020 ± 2268760 ± 3558980 ± 3929640 ± 4433734 ± 2183786 ± 2433667 ± 212
44248 ± 176 a2972 ± 119 b2475 ± 194 b8640 ± 461 a6460 ± 314 b6760 ± 284 ab3600 ± 3023976 ± 2513886 ± 282
64202 ± 213 a2442 ± 132 b1235 ± 107 c8320 ± 409 a6080 ± 206 b6240 ± 241 b3610 ± 2253875 ± 2273742 ± 229
113740 ± 160 a1998 ± 118 b1070 ± 69 c7234 ± 377 a4864 ± 288 b4622 ± 250 b3443 ± 237 a2794 ± 184 ab2061 ± 230 b
Table 4. Dynamics of changes in the additional biochemical parameters of the FS incubation process in control and experimental (with A. faecalis) tanks. Means ± standard deviations (SD) are given. Different letters reveal a significant difference from post hoc Tukey’s test analysis.
Table 4. Dynamics of changes in the additional biochemical parameters of the FS incubation process in control and experimental (with A. faecalis) tanks. Means ± standard deviations (SD) are given. Different letters reveal a significant difference from post hoc Tukey’s test analysis.
Incubation Time, DaypHNtot, mg/L N-NH4, mg/LBicarbonate Ion, mg/L
Tank1Tank2Tank3Tank1Tank2Tank3Tank1Tank2Tank3Tank1Tank2Tank3
09.549.559.563846 ± 1933869 ± 2363715 ± 2283218 ± 2683254 ± 2053234 ± 2105000 ± 2065035 ± 2354916 ± 199
19.579.629.533716 ± 2063609 ± 2333571 ± 2183424 ± 1983513 ± 1923531 ± 2214734 ± 1674368 ± 1783941 ± 191
49.549.619.573123 ± 2152957 ± 1592876 ± 1412857 ± 1362851 ± 1462790 ± 1714606 ± 1594180 ± 1633862 ± 161
69.629.639.582939 ± 1962834 ± 1652766 ± 2072674 ± 1142760 ± 1082667 ± 1594553 ± 200 a3964 ± 185 ab3024 ± 156 b
119.649.609.542818 ± 1742790 ± 2272692 ± 1152541 ± 1412697 ± 1732613 ± 1443769 ± 188 a3073 ± 187 ab2683 ± 142 b
Table 5. Changes in cell titre and coliform counts during incubation. Means ± standard deviations (SD) are given. Different letters reveal a significant difference from post hoc Tukey’s test analysis.
Table 5. Changes in cell titre and coliform counts during incubation. Means ± standard deviations (SD) are given. Different letters reveal a significant difference from post hoc Tukey’s test analysis.
Incubation Time, DayTank1Tank2Tank3
Total Cell TitreColiform PercentageTotal Cell TitreColiform PercentageTotal Cell TitreColiform Percentage
Starting point
(40 min)		(2.9 ± 0.3) × 106 a30.5(4.3 ± 0.1) × 106 a29.4(8.0 ± 0.5) × 106 b28.6
1(4.0 ± 0.3) × 10626.5(4.4 ± 0.4) × 1068.6(4.1 ± 0.2) × 1067.4
4(4.6 ± 0.3) × 106 a20.8 (7.3 ± 0.5) × 106 b0(8.1 ± 0.4) × 106 b0
6(6.0 ± 0.5) × 105 a17.2(1.3 ± 0.1) × 107 b0(8.4 ± 0.7) × 106 c0
11(2.7 ± 0.3) × 104 a13.9(9.1 ± 0.8) × 106 b0(1.2 ± 0.1) × 107 b0
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Loiko, N.; Kanunnikov, O.; Litti, Y. Use of Alcaligenes faecalis to Reduce Coliforms and Enhance the Stabilization of Faecal Sludge. Sustainability 2023, 15, 12580. https://doi.org/10.3390/su151612580

AMA Style

Loiko N, Kanunnikov O, Litti Y. Use of Alcaligenes faecalis to Reduce Coliforms and Enhance the Stabilization of Faecal Sludge. Sustainability. 2023; 15(16):12580. https://doi.org/10.3390/su151612580

Chicago/Turabian Style

Loiko, Nataliya, Oleg Kanunnikov, and Yuriy Litti. 2023. "Use of Alcaligenes faecalis to Reduce Coliforms and Enhance the Stabilization of Faecal Sludge" Sustainability 15, no. 16: 12580. https://doi.org/10.3390/su151612580

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