The Development of Anammox and Chloroflexi Bacteria during the Composting of Sewage Sludge

Abstract

The C/N ratio is an extremely important parameter in the composting process and is directly responsible for the growth of microorganisms. A low C/N ratio contributes to higher emissions of greenhouse gases and odorous substances, such as ammonia (NH₃), which is formed by nitrogen mineralization. Due to the highly toxic effects of ammonia, it is a particularly unwanted by-product that can disrupt the composting process since it poisons microorganisms and causes environmental issues. The activity of anammox bacteria, so far analyzed only in wastewater treatment processes, is a particularly efficient method of nitrogen removal, having an advantage over the conventional methods used previously. This study shows the presence of anammox bacteria during composting, which allows for an opportunity to improve the process and reduce its impact on atmospheric pollution. Despite the aerobic nature of this process, the composted mass of waste presents conditions conducive to the development of these ammonia-oxidizing bacteria, as well as the other strains of microorganisms cooperating with them. This makes it possible to compost at a low C/N ratio; in addition, there is no need for an additional energy supply through aeration, as the processes carried out by anammox bacteria do not require oxygen. Therefore, more in-depth research is necessary to evaluate the low C/N effect on anammox and Chloroflexi bacteria growth and its effect on nitrogen balance during composting.

1. Introduction

Composting is a microbiological process which involves the biochemical conversion of organic matter into humic substances for use in agriculture as soil fertilizers [1]. This complex physical and chemical process is characterized by a dynamic course. The most important parameters that affect the process are temperature, pH, presence of microorganisms, aeration, and the C/N ratio [2]. In the composting process, there is a strong relationship between these; at the same time, very often, the biochemical processes that occur in the compost pile experience changes in the ambient conditions, the composition of the composted material, and the microbial community inside [3].

The C/N ratio is an extremely important parameter in the composting process, directly responsible for the growth of microorganisms. Essential findings about composting indicate that the optimum C/N value for composting material that contains organic compounds susceptible to biological oxidation is 20 ÷ 35 [4,5,6]. A low C/N ratio contributes to higher emissions of greenhouse gases and odorous substances, such as ammonia (NH₃), which is formed by nitrogen mineralization. Due to the highly toxic effects of ammonia, it is a particularly unwanted by-product that can disrupt the composting process since it poisons microorganisms and causes environmental issues [2,7]. If the C/N ratio is too high, the decomposition of organic matter in the compost slows down. The reason for the slowdown in decomposition is a reduction in the activity and development of the microorganisms [8].

Biological decomposition involves a sequence of complex biochemical reactions in the presence of catalysts (enzymes), resulting in the partial conversion of organic compounds into so-called humus during the humification process and their partial oxidation to mineral compounds (such as CO₂, H₂O, NH₃, NO_x) in the mineralization process. The final effect of humification and mineralization makes the organic matter decomposition method applicable in the transformation of municipal waste containing high amounts of compounds susceptible to biological decomposition [1].

Enzymes produced by bacteria metabolism are crucial in the process of nitrogen transformation during composting. These transformations include several processes, such as ammonification, nitrification, anaerobic ammonia oxidation, and denitrification [9]. The direction and sequence of nitrogen transformation during composting can be presented in the following way:

NH₃ (gas) ↔ NH₃ (aq) ↔ NH₄⁺ ←organic nitrogen→NH₄⁺ →NO₂⁻ ↔No₃⁻

(1)

These processes are fundamental in the global nitrogen cycle and must be sustained in engineered and natural environments, including those with extreme conditions. Currently, ammonification, nitrification, and denitrification are the three main processes considered for measures to prevent N loss in composting, yet anaerobic ammonia oxidation (anammox) has recently been recognized as an alternative microbial metabolic pathway in the denitrification involved in N₂ emissions [10]; therefore, it is seen as the most important way to reduce N loss during low C/N composting.

In the composting process, high temperatures (~70 °C) play a crucial role in the reduction of pathogens [2], but such thermal conditions can be harmful for anammox bacteria [11]. In this part of the composting process, Chloroflexi, as a putative nitrite-oxidizing bacteria, is significantly important in the nitrification process (nitrite oxidation) [12].

There are several related scientific literatures describing the composting of mixtures of waste within the broad scope of the C/N = 15 ÷ 28.9 ratio [8,13,14], but none explain the a process that avoids the risk of environmental harmful emission and loss of nitrogen in the final product. Additionally, the composition and dynamics of nitrifier and denitrifier communities in compost have been analyzed in many studies, but only a few confirmed the presence of anammox [15,16] or Chloroflexi bacteria [12,17,18]. The nitrogen transformation process in composting is reported as a complex one, including several pathways such as ammonification, nitrification, denitrification, and anaerobic ammonia oxidation; with this, it is crucial to understand the mechanisms of the last process, conducted by anammox bacteria [1,2] as a potential source of the future regulation mechanism of NH₃ emission from composting. Anammox bacteria have been found in different ecosystems, including stream sediments, wetlands, and groundwater [3,4,5], but only a few sources described the potential participation of these microorganisms during aerobic waste treatment processes. The lack of research in this area makes it impossible to determine a quantitative link between functional gene groups and nitrogen transformation, and consequently, its phenomenal change during composting remains an unexplored gap [2]. Meanwhile, when conducting an analysis on the presence of anammox bacteria in the composting process, it is a necessary alternative to refer to the wastewater treatment and anaerobic processes for which research has been carried out more frequently and for a broader scope.

This study focused on the evaluation of the presence of microbial communities during the composting of the substrate (sewage sludge) with a low C/N ratio. Particular emphasis was given to anammox bacteria and environmental conditions that affect their role in nitrogen balance during the process. This research opens up new possibilities in the field of composting process control, especially the process carried out in difficult processing conditions and the control of harmful gases such as NH₃ or N₂O in the future.

2. Materials and Methods

2.1. Material Characteristic and Experiment Configuration

Field research was performed at the wastewater treatment plant in Goleniów (West Pomeranian Voivodeship, Poland). The treatment plant usually produces ca. 5500 Mg/y of mechanically dehydrated sewage sludge. Composting was carried out in roofed piles, approximately 70 m long with a trapezoidal transversal cross-section. The cross section had dimensions of a 3 m base width, 1.5 m height, and A m top width. The piles were mechanically overturned. In the first three weeks, overturning was done twice a week and once in the succeeding weeks. Composting takes four–five months, depending on the external conditions. The composting process was carried out in closed aerated reactors. After this period, the compost was used for agricultural purposes [1].

The samples were collected from the composting piles on the 1st week, after 2 weeks, and after the 4th week of composting, and were labeled as samples A, B, and C, respectively (Figure 1). For each sample, basic analysis was carried out: pH, moisture content (MC), volatile solids (VS), elemental composition (C, H, N content), C/N ratio, and microbial community analysis via PCR analysis. During the composting process, the changes in temperature in all piles were monitored.

2.2. Methods

2.2.1. Determination of the Physicochemical Properties of Compost Samples

The samples (A, B, C) were subjected to drying at 105 °C until a constant mass was achieved and was used as a basis for the determination of the moisture contents. Further heating of the samples at 550 °C in a muffle furnace to determine the volatile solids (VC) was carried out. Elemental compositions of C, H, and N were determined according to PN-EN ISO 11885:2009, with the use of a ICP-AES iCAP 7400 Thermo Scientific spectrometer.

2.2.2. Microbiology Community Analysis

Fresh composting samples were collected after the manual turning of the composting materials to track the dynamics of microbial community structures. Briefly, this analytical method involved DNA extraction using GeneMatrix Environmental DNA/RNA Extraction kit (Eurx, Gdańsk, Poland) and the amplification of the V3–V4 hypervariable region of bacterial 16S rRNA gene, with primer pairs of:

• 16S_V3-F 357F: (5-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-CCTACGGGNGGCWGCAG-3) and;
• 16S_V4-R785R: (5-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-GACTACHVGGGTATCTAATCC-3).

This was carried out by polymerase chain reaction (PCR) and high-throughput sequencing on the Kapa HiFi PCR Mix (Roche) [19]. The extracted DNA was processed via 1% agarose gel electrophoresis using a spectrophotometer Qubit 3.0 (Thermo Scientific, Waltham, MA, USA) to evaluate the integrity, purity, and concentration.

The reaction was carried out using 25 μL volumes, containing 2.5 μL (5 ng/μL) genomic DNA, 5 μL (1 μM) of each primer, and 12.5 μL of 2× KAPA HiFi PCR Mix (Roche). PCR reaction mix contained 2.5 μL distilled water instead of genomic DNA, as a negative control. Amplification was performed using the following program: initial denaturation at 95 °C for 2 min, followed by 25 cycles of 95 °C for 15 s, 55 °C for 10 s, and 72 °C for 30 s, and a final extension at 72 °C for 7 min before cooling to 10 °C. PCR products (∼450 bp) were purified with Agencourt AMPure XP beads (Beckman Coulter genomics) to remove free primers and primer dimer species.

Next generation sequencing was carried out using Illumina MiSeq platform utilizing MiSeq Reagent kit v3 (600 cycles). Sequencing of amplicons with the MiSeq engine resulted in the following amounts of data (number of paired Illumina raw readings):

• Sample A–95608;
• Samples B–95152;
• Sample C–125302.

The number of readings obtained after the filtering procedure was sufficient to carry out further stages of the analysis. It was assumed to obtain a min. of 15,000 paired readings for each test run. The readings obtained were filtered using the fastp tool (github.com/OpenGene/fastp (accessed on 4 June 2022))–the remaining sequences of the sequencing adapters and the low-quality readings were removed. The sequences with ≥97% similarity were all gathered into operational taxonomic units (OTUs) by usearch software (V10). Alpha diversity of bacterial community (i.e., Observed species, Chao1 and Shannon index) and Beta diversity were calculated with QIIME2. After quality filtering, sequences with ≥97% similarity were assigned to the same OTU to represent a species, which were annotated for taxonomic information (set the confidence threshold to default to ≥0.5) to get the OTU taxonomy synthesis information table for the final analysis using silva (https://www.arb-silva.de/ (accessed on 3 June 2022)) database [20].

The VOS viewer software, with notepad as editor, was used to create a network map among the syntropic bacteria from the study. The relative abundance of each bacterium served as a basis for the occurrence and link in the map. Tab-delimited files, imported in notepad, were used to prepare the code for all bacteria, including those with a relatively low abundance, serving as both a source and type for the bibliographic data of the Web of Science. Further, the type of analysis employed was based on the cooccurrence of bacteria. Full counting with one as the threshold for the minimum occurrence of each bacterium was more appropriate in the generation of the network map. The circle represents each bacterium and the size indicate their occurrence, which was based on the relative abundance, whereas the curved line was chosen to indicate the link between the bacteria and the relative size represented links. Cluster coloring was used to distinguish the groups of bacteria that were most abundant, slightly abundant, and those that were not abundant.

3. Results and Discussion

3.1. Compost Characteristics and Temperature Changes during the Process

The composting samples used in these experiments had a pH of 8.93–8.99, an MC of 63.1–66.7%, and a TS of 77.4–84.0%, and were characterized by 34–44%, 4.8–6.3, 1.1% C, H, N, respectively (by dry mass base) (

Table 1. Properties of composting samples.

Sample	Moisture Content, %	Volatile Solids, % d. m.	pH	C, %	H, %	N, %	C/N
A	66.3	78.7 ± 3.3	8.93	34 ± 7	4.8 ± 1.0	1.1 ± 0.2	36.3
B	63.1	77.4 ± 1.8	8.98	40 ± 8	5.8 ± 1.2	1.1 ± 0.2	43.6
C	66.7	84.0 ± 1.6	8.99	44 ± 9	6.3 ± 1.3	1.2 ± 0.2	42.7

). Such results are typical for the composting process [3]. The addition of wood chips significantly changed the C/N ratio in the sewage sludge from 5–10 [1,21] to 36–44. The use of woodchips helped to improve the C/N ratio but also gave the structure necessary in the composting process to provide air into the pile. However, the C and N from the wood chips did not contribute to the composting process due to low biodegradability. Therefore, the realistic C/N ratio should be considered for raw sewage sludge ~5 [1]. Even in a system with good aeration and structure, there are still possibilities for places with low oxygen and high-temperature hotspots and places with a low temperature [22]. Such places are an ideal environment for growing anammox bacteria (coldspots [16]) or Chloroflexi (hotspots [18]).

During the process, the internal temperature was measured (Figure 2). The information on temperature during the sampling process is highlighted using a red line. It was observed that in each place, thermophilic temperatures were dominant (>50 °C).

3.2. Community of Bacteria Structure Changes

Few studies that investigate the presence of anammox bacteria in the composted mass of waste show that their activity is characteristic of the thermophilic phase of the process [6]. The reports of Byrne et al. and Rysgaard et al. [7,9] agree with this, according to which these bacteria are characterized by high resistance to functioning in extreme environments where the temperature exceeds 85 °C or is lower than −2 °C. As presented by Robledo-Mahon, et al., these conditions become an interesting ecosystem for fungi and bacteria able to resist high temperatures, where a total of 105 fungal strains and 128 bacterial strains were initially isolated [23].

This is in line with the results presented in this paper; the temperature of the composting process during the collection of each sample was in the thermophilic range (~50–70 °C, Figure 2), with a greater diversity of anammox bacterial groups found under these conditions [6] in connection with the availability of nitrogen compounds. According to the authors, the precondition for the presence of these microorganisms is the coexistence of ammonium and nitrite nitrogen under anaerobic conditions; the concentration of the former increases precisely during the thermophilic phase of composting. In turn, the source of nitrite for these microorganisms is the nitrification process, the activity of which was recorded at temperatures close to 70 °C [10]. Additionally, due to the progressive decomposition of simple organic matter, which, in turn, results in a rapid increase in temperature and oxygen depletion, anaerobic conditions may arise in this phase of the composting process, favoring the growth of anammox bacteria.

However, it was reported that in anammox reactors, in addition to typical anammox bacteria, there are microorganisms that interact with each other through enzymatic processes, creating an environment conducive to anaerobic ammonia oxidation processes. Figure 3, prepared in VOSviewer software, shows the syntropic relationship, through the link lines, among the microorganisms reported in the research at the phylum level. The size of the circle represents the relative abundance of each bacterium. Those that are connected through the lines are considered syntrophic bacteria as they were relatively abundant and were enriched throughout the composting operation. Other bacteria, not linked in the map, were only detected at the end of the decomposition process with a relative abundance lower than 1.0% (Acidobacteria, Armatimonadota, Cyanobacteria, Thermogota, and wps-2), and for this reason, they are not considered as a part of syntropy of the community.

In the early stage of the composting process (sample A), Firmicutes were the most abundant, constituting around 48% of the total population, followed by Proteobacteria (16.8%), Actinobacteria (14%), and Bacteroidota (12%, Figure 4). Similar bacterial groups were detected in the sludges after mesophilic anaerobic digestion, where the phylum Bacteroidetes, Chloroflexi, Firmicutes, and Proteobacteria were predominant; this suggests that the composting process allows this bacterium to survive even when the environmental conditions change [24].

In comparison, at the later stages of decomposition (sample B), Chloroflexi showed the highest percentage increase of 2068% (from 0.04% to 1.1%), followed by Deinococcota (1352.7%) and Myxococcota (603%). Firmicutes and Proteobacteria were slightly suppressed toward the end of the decomposition (sample C). The presence of these groups is also confirmed by other authors; in each of the analyzed sludge samples by Chen et al., the microorganisms discovered came from the major phyla of Proteobacteria, Chloroflexi, Planctomycetes, and Bacteroidetes [12]. In addition, the authors also noted the increase in the Chloroflexi community with the increase of the duration of the process; they explained this situation by the fact that the development of these groups is based on the use of cellular compounds derived from already dead microorganisms and their metabolites [13]. So far, it has been reported that the Anaerolineae and Caldilineae classes were distinguished from among the Chloroflexi present in anammox reactors [15,16,17], of which representatives of the former were also observed in the studies described here (Supplementary Material Table S1). A similar role to Chloroflexi was also discovered for Bacteroidota, which, in the studies described here, were the fourth most numerous group of microorganisms in the composting process. However, the role of these microorganisms in anammox reactors has not been fully explored, although the studies conducted so far have shown that they possess genes responsible for nitrogen binding; thus, they act in the nitrogen cycle and may even be able to bind to N₂ [17]. Additionally, Robledo-Mahom et al. proved that a high percentage of isolates bacterium from sewage sludge composting are able to produce enzymes, such as polyphenol oxidase, peroxidase, amylase, and ammonifying enzymes, confirming the ability of ammonium reduction from the process in a good environment [23]. The practical information is that the growth of anammox bacteria can reduce ammonium gas emission and N losses and can be effective yet cheaper in comparison to the addition of, for example, zeolite to compost to obtain the same results [25].

However, it is worth emphasizing that they can also have a toxic effect on anammox bacteria due to their phosphate removal; additionally, they compete with each other in terms of the denitrification of nitrite [19,20]. Therefore, it is possible that anammox bacteria were not present in the analyzed compost samples due to their earlier elimination by the Bacteroidota noted here. An additional argument here is the fact that they can work in both aerobic and anaerobic conditions, thus gaining an advantage in the process of aerobic waste treatment [21].

It is also worth mentioning that the genera of anammox bacteria found so far were described in the literature as belonging to Planctomycetes. In this study, the presence of five representatives of this phylum was noted (orders Pirellulales, Isosphaerales, Tepidisphaerales, Planctomycetales, Supplementary Material Table S1). Although the order Brocadiales, into which the currently known anammox bacteria are classified, was not discovered here, there is an indication that such bacteria could be present at an earlier stage of the process and then, after the composted mass reached unfavorable conditions (temperature exceeding the optimal value of 40 °C), they were lysed; related microorganisms from the same phylum may have appeared in their place. The pH of the composted waste may also be included in the conditions unfavorable for the further development of anammox bacteria. The samples analyzed here were characterized by an alkaline pH close to 9, while the ideal pH range for them was 6.7–8.3 [26]. However, this could be beneficial since the results presented by Malcheva et al. showed that the creation of an alkaline environment led to a reduction in the number of beneficial microorganisms. This can be used to reduce or destroy pathogenic microorganisms in sewage sludge, compost, and organic waste [27].

Considering the large group of Proteobacteria in the analyzed samples, it should be noted that two classes of microorganisms dominated: Alphaproteobacteria and Gammaproteobacteria, for which 18 and 16 representatives were identified, respectively (Figure 4). A similar observation was made by Gao et al. [28]. During their analysis of the role of long- and short-chain N-acyl-l-homoserine lactones on microbial community dynamics in activated sludge, they distinguished various groups of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) participating in the ammoniacal nitrogen removal process and indicated Alphaproteobacteria and Gammaproteobacteria were the main representatives.

One of the factors limiting the use of anammox bacteria in the treatment of sewage sludge is their slow growth. Due to their long doubling time, they are susceptible to external factors, such as the previously mentioned complex ecosystem, the introduction of other microorganisms that are their competitors, or sudden changes in the conditions prevailing in the reactor, e.g., during accidental leaching of sediments [29]. The doubling times of anammox bacteria range from 1.8 to 11 days [30], and the associated delays in reactor start-ups were noticed by other researchers [31]. A similar effect was observed in the experiment described here. For many microorganisms from the predominant sample’s phyla, Proteobacteria, Bacteroidota, Chloroflexi, Acidobacteriota and Planctomycetota, their highest abundance was recorded in the second week of composting, 14 days after starting the process (in case of 16, 7, 5, 2, and 1 families, respectively, Supplementary Material Table S1). On the other hand, a lower presence of these bacteria in the samples taken after the fourth week can be explained by the competition effect, as well as the gradual depletion of substances necessary for their dynamics.

4. Conclusions

The activity of anammox bacteria, so far analyzed only in wastewater treatment processes, is considered to be a particularly efficient method of nitrogen removal, having an advantage over the conventional methods used previously. The benefits of using them are not only operational, but also environmental. Based on the analyses carried out so far, it has been shown that in addition to reducing the costs of the wastewater treatment process by up to 90%, the necessary infrastructure and space are also reduced. Considering the environmental aspects, an important element is the lack of production of additional CO₂, which is simultaneously used by anammox bacteria, preventing the process from contributing to an increase in the amount of greenhouse gases.

The discovery of the presence of anammox bacteria during composting becomes not only a new research issue, but also an opportunity to improve the process and reduce its impact on atmospheric pollution. Despite the aerobic nature of this process, the composted mass of waste presents conditions conducive to the development of these ammonia-oxidizing bacteria, as well as other strains of microorganisms cooperating with them. This makes it possible to compost at a low C/N ratio; in addition, there is no need for an additional energy supply through aeration, as the processes carried out by anammox bacteria do not require oxygen.

However, the preliminary research carried out so far does not fully explain this phenomenon. It is necessary to implement experiments that explain the mechanisms of anammox bacteria activity in the composting process, describing the optimal conditions for their development and considering the further effectiveness of the process and the production of a valuable end-product. For this purpose, it is necessary to simultaneously monitor the growth of these bacteria and the processes of ammonia oxidation and nitrite reduction, as well as the use of molecular biology techniques.

The presented preliminary research is represented by only three samples, taken during different stages of composting; yet, these preliminary research findings may open a new niche for the investigation of the composting process and therefore, it should be continued.