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Article

The Impact of Chicken Manure Biochar on Antibiotic Resistance Genes in Chicken Manure Composting

by
Kamalya Karamova
,
Natalia Danilova
,
Svetlana Selivanovskaya
and
Polina Galitskaya
*
Institute of Environmental Sciences, Kazan Federal University, 420008 Kazan, Russia
*
Author to whom correspondence should be addressed.
Agriculture 2022, 12(8), 1158; https://doi.org/10.3390/agriculture12081158
Submission received: 22 June 2022 / Revised: 29 July 2022 / Accepted: 30 July 2022 / Published: 4 August 2022
(This article belongs to the Special Issue Long Term Agronomical, Physiological and Environmental Implications of Biochar Use in Agricultural Soils)
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Abstract

:
One way to increase yields in agriculture using organic alternative methods is the introduction of manure-based composts into the soil. However, the use of such composts carries a risk of soil contamination with antibiotic resistance genes (ARG) from the gut and manure of the livestock. The contamination of the composts with heavy metals or antibiotics can increase this risk, while the addition of porous materials, such as biochar, to the composts has the potential to decrease it. This study is devoted to revealing the fate of ARGs in bedding chicken manure composted with the addition of oxytetracycline (OTC), heavy metals, and chicken manure biochar. It was revealed that the additives did not affect the physicochemical parameters of the compost. The bacterial communities in different composting mixtures had similar structures and dynamics. It was revealed that the shifts of the bacterial compositions of the composting mixtures were mainly determined by the duration of the process. However, some minor differences in the OTU (operational taxonomic unit) levels were observed between the variants. The addition of biochar and metals led to 26.7% and 34.5% decreases, respectively, in the number of tet(A) gene copies, while the addition of oxytetracycline led to a 43.7% increase. The number of copies of the int1 gene increased by 45.9% after the addition of oxytetracycline. The correlation between the abundance levels of different bacterial OTU and ARG contents was estimated, and biochar’s impact on those OTUs was analyzed. It was assumed that some OTUs might be carriers of ARGs (such as Natronobacillus, Luteimonas, and Trichococcus), and their abundance in the presence of the biochar decreased due to competitive exclusion by noncarriers (such as Corynebacterium, Clostridia, and Halorhodospira). The use of biochar in composting can be considered a way to reduce the contamination of the final composts with ARGs.

1. Introduction

The quality of agricultural soil is very important because it is directly related to its fertility and the plant yield, which in turn is directly related to global food security. Many studies have demonstrated that the use of mineral fertilizers contributes to an increase in yields but leads to a drop in soil quality in the long term [1]. Therefore, alternative methods of fertilization have been discussed, with the use of livestock manure compost being one of them [2]. The use of compost as an organic soil fertilizer remains an integral practice in agriculture, as it improves the physicochemical and biological parameters and supplies plants with important nutrients, thereby increasing yields [3]. However, the manure from large-scale farms often contains antibiotics. These are antibacterial compounds that are widely used in veterinary and livestock husbandry in order to heal and prevent diseases and in order to stimulate livestock weight gain. Chicken meat is the main source of protein for the Earth’s population, and the amount of antibiotics used in chicken husbandry exceeds the amounts used for other livestock types [4]. The most commonly used antibiotics in chicken husbandry are tetracyclines, sulfonamides, and macrolides [5,6].
Manure is usually contaminated by antibiotic compounds and their metabolites, since only a low proportion of antibiotics is absorbed in the chicken gut [5,6]. Antibiotics can enter the soil from the compost and can negatively affect plant yields, since they inhibit the functioning of earthworms, reduce the diversity of bacteria, increase the proportion of plant pathogenic species, and reduce the bioavailability of nutrients [7].
Bacteria are able to quickly develop mechanisms to resist antibiotics. Therefore, manure and manure-based composts contain various microbes carrying antibiotic resistance genes (ARGs) able to be transferred to other generations through vertical transfer or other populations through horizontal transfer [5]. The problem of the ARG contamination of manures and fertilizers made of these manures is considered even bigger than the original problem of antibiotic contamination, since the resistance may be transferred and can last for months or even years, while antibiotic compounds usually decompose within several days or weeks [5,6]. The existing risks of ARG spread in soils and plants should be considered, as a threat to public health could be created by the consumption of agricultural products [8].
There are several mechanisms by which microorganisms have developed resistance to antibiotics. Those mechanisms can be encoded by one or several specific genes located in chromosomes or more often in plasmids [9]. In the case of tetracyclines, which are the most widespread antibiotics in poultry husbandry, about 40 different genes encoding resistance have been revealed. For example, the genes tet(A), tet(B), tet(C), tet(D), tet(E), tet(G), tet(H), tet(I), tet(J), and tet(Z) encode the active antibiotic efflux. The genes tet(M), tet(O), tet(S), tet(W), tet(Q), and tet(T) encode ribosome protection, and the tet(X) gene encodes antibiotic detoxification [10]. Bacteria may possess several ARGs encoding different ways to resist one and the same antibiotic compound, or to resist different antibiotics [11]. Many reports have demonstrated that composting leads to a decrease in the ARG content in manure, up to their total removal [12,13,14]. Some reports have underlined only a temporary decrease in ARG copy numbers in the thermophilic phase of composting, with regeneration in the following phases [15,16]. Rare reports have even demonstrated an increase in ARG copy numbers during the process of composting [17,18]. Those contradictory results were observed due to the complexity of the processes occurring during composting: on the one hand, ARG-carrying microbes might be eliminated in the thermophilic phase of composting, since they are not tolerant to high temperatures; on the other hand, the elevated temperatures might intensify the horizontal gene transfer and enlarge the number of ARG-carrying species [19]. Additional factors such as the manure origin, its level of contamination by antibiotics and AGRs, the type of antibiotics, the temperature and duration of the thermophilic phase, the biogenic elements, the moisture and other abiotic parameters involved in composting, and the presence of specific compounds such as heavy metals that are known to be intensifiers of ARG horizontal transfer may play significant roles in the fate of ARGs in the final compost [14,20,21,22,23]. Therefore, additional technologies for the improvement of ARG removal from compost are required. One of those is the addition of a porous material such as a zeolite or biochar to the composting mixture [24]. It is believed that the porous material may absorb antibiotic compounds, mitigating their selective pressure; absorb mobile genetic elements (plasmids, integrons, and transposases), mediating the horizontal gene transfer of ARGs; and create favorable conditions for some populations, causing bacterial succession in the direction of a nonresistant community [14,25,26,27,28]. However, the mechanisms of biochar influence on the compost bacterial community, including the ARG carriers, are not fully clear yet. In addition, the introduction of biochar into the soil as part of the compost can increase the plant biomass, and accordingly can contribute to the accumulation of organic matter in the soil, since its content is closely related to nutrient cycling, soil quality, and fertility. Previously, the best results have been obtained for biochar made of plant biomass, such as straw or wood [14,27,29,30,31,32,33]. Biochar made of chicken manure has high potential as a soil fertilizer, since it possesses not only common biochar characteristics such as high porosity and a large surface area, but also specific characteristics originating from the initial substrate—high nitrogen and phosphorous contents [34]. In the literature, data concerning this biochar type’s influence on the processes of chicken manure composting, including the removal or enrichment of ARGs and the mechanisms of this process, are presented [34].
Based on our limited understanding of the fate of ARGs in chicken manure compost mixtures amended with chicken manure biochar, the overall objective of this study was to reveal the factors most affecting the genes tet(A) and tet(X) during the process of chicken manure composting. The genes tet(A) and tet(X) encode two different mechanisms of resistance of bacteria to tetracyclines, which are the most widespread group of antibiotics in poultry husbandry. For this, we composted chicken manure with and without biochar additions, as well as with and without the addition of oxytetracycline and heavy metals. The physicochemical parameters, as well as the respiration rates, ARGs and integron class 1 gene copy numbers, and the structure of the bacterial community, were monitored during the process of composting.

2. Materials and Methods

Bedding chicken manure was obtained from the poultry farm “Chelny-Broiler” (Naberezhnye Chelny, Tatarstan Republic). This is one of the 10 largest chicken farms in Russia, producing about 150,000 t chicken manure per year. The following variants of compost mixtures were prepared: control K (without additives), B (with the addition of 15% biochar, w/w), O (with the addition of the antibiotic oxytetracycline at a concentration of 300 mg kg−1), M (with the addition of metals Ni, Cd, Fe, and Cu at concentrations of 70, 130, 1500, and 1000 mg kg−1), MO (with the addition of metals and the antibiotic), and their combinations (variants BO, BM, and BMO). Since the presence of heavy metals is able to intensify the horizonal transfer of ARGs, and the purpose of the present study was to reveal the fate of ARGs in the composting mixtures, variants with heavy metals (raw and with the addition of biochar) were used in this study.
All composting variants were prepared in three replicates. Each replicate included 60 kg of raw bedding chicken manure and 18 kg of straw, which was used as a structuring agent. Some replicates contained additives (biochar, heavy metals, and oxytetracycline) in the amounts mentioned above. The composting was carried out for 120 days in rotary drum composters with a volume of 200 L with daily stirring for 1 h at a speed of 40 rpm (Supplementary Figure S1. The temperature in the compost mixtures was measured daily. On the 1st, 3rd, 7th, 14th, 21st, 28th, 35th, and 42nd days, and then once every two weeks, samples were taken for the analysis of the dissolved organic carbon; the respiration activity; the numbers of bacteria, fungi, and antibiotic-resistant genes; the int1 gene; and the structure of the microbial community. All measurements were carried out in three replicates.
The content of dissolved organic carbon was estimated using the wet oxidation method in accordance with ISO 14235:1998, using the process of oxidation of chromium ions Cr6+ to Cr3+ with pre-extraction with an aqueous solution of potassium sulfate [35]. The respiration activity of the microorganisms was evaluated based on the amount of carbon dioxide released during the incubation in hermetically sealed vessels according to ISO 16072:2002 [36].
The extraction of DNA from samples was performed immediately after sampling using a FastDNA™ Spin Kit for soil DNA (MP Biomedicals, Irvine, CA, USA). The resulting nucleic acid was used for the real-time PCR to assess the content of ARGs (tet(A), tet(X)), the int1 gene, and the total copy numbers of bacteria and fungi [37]. The structure of the bacterial community was assessed on the basis of 16S amplicon sequencing using the Illumina MiSeq method. To do this, the total DNA isolated from the samples was purified using the QIAquick PCR Purification Kit (Qiagen, Düsseldorf, Germany). The DNA concentration was determined on a Qubit Fluorometer (Invitrogen, Waltham, MA, USA) using a Quant-iTds DNA High-Sensitivity Assay Kit (Thermo Fisher, Waltham, MA, USA). The samples were further prepared using the MiSeq Reagen Kit v2 and a MiSeq device (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. The obtained sequence data were analyzed using the QIIME program [38]. The chimeras were excluded from analysis using the usearch61 algorithm. The bacterial operational taxonomic units (OTUs) were determined according to the Greengenes database. The taxonomic classification was performed using the RDP classifier and PyNAST [39]. The community diversity analysis was performed using the R vegan software package (http://sortie-admin.readyhosting.com/lme/R%20Packages/vegan.pdf, accessed on 5 November 2020).
All studies were carried out at least in triplicate. The statistical processing of the obtained results was carried out using Microsoft Office Excel 2010 (Redmond, WA, USA). All tabular and graphical data contain average values and standard errors. To assess the significance of differences, Fisher’s test was used at α = 0.05. The beta diversity was calculated using the R software package (www.r-project.org, accessed on 5 November 2020). Multivariate scaling based on the Bray–Curtis coefficient was used to determine the beta diversity [40]. The mathematical modeling (robust linear model) and correlative analysis (RandomForest method) were carried out in the R software package (www.r-project.org, accessed on 5 November 2020).

3. Results

3.1. Physicochemical Parameters of Composting and Respiration Activity of Compost Mixtures

The results of the assessment of the temperature, dissolved organic carbon, and respiration activity are shown in Figure 1a–c. The temperature profiles of the compost mixtures generally coincided for all variants and did not depend on the addition of biochar, oxytetracycline, or heavy metals. Interestingly, a slight short-term increase in temperature was observed between days 2 and 5 in the variants B, BO, BM, and BMO, and between days 9 and 13 in the variants B and BO. Since this effect was observed only for the variants containing biochar, it can be assumed that the biochar played a role in the intensification of the microbes, enabling exothermic processes of decomposition of organic matter in the composting mixtures.
The dynamics patterns of the contents of dissolved organic carbon were alike for all variants of compost mixtures (Figure 1b). On the 1st day, the values were about 5–6 mg g−1, then by the 14th day a sharp increase up to 21–32 mg g−1 was observed. The dissolved organic carbon levels remained high until the 21st day. Later on, a decrease to 2–5 mg g−1 was recorded. Starting on the 35th day, there were no significant changes in the content of dissolved organic carbon. All compost mixtures also showed similar dynamics of respiration activity during the entire composting process. On the 1st day, the values ranged from 0.002 to 0.008 mg CO2 g h−1. Furthermore, by the 3rd day, in all variants there was a sharp increase in the level of respiration to 0.008–0.013 mg CO2 g h−1, which coincided with the thermophilic phase of composting. Interestingly, the highest values for the respiration activity were noted for those variants of compost mixtures that contained the antibiotic and heavy metals (all samples except the control and B samples).
Thus, the addition of the biochar, antibiotics, and heavy metals to the compost mixtures did not generally have a significant effect on the physicochemical parameters during composting; however, it somehow affected the level of respiration of the composts.

3.2. Bacterial and Fungal Counts, and Copy Numbers of tet(A), tet(X), and int1 Genes

Figure 2a,b gives data on the number of copies of genes, reflecting the amounts of bacteria and fungi, on the 1st and 84th days of the experiment. On the 1st day, the bacterial gene copy numbers did not differ between the samples and ranged between 1.16 and 2.21 × 108 copies g−1. By the end of the composting, the numbers of bacterial copies of genes decreased in all samples by 7–20-fold, and significant differences were observed between the numbers of samples; for example, the minimum value for this parameter was found in the BO sample (6.25 106 gene copies g−1), which differed significantly from that of samples O, M, MO, and BMO. In the fungal community, on the contrary, a decrease in the number of copies of genes was not detected for any of the samples, while in the samples of BO, BM, and BMO there was a slight but statistically significant increase in this indicator (from 2.6 × 104–4.28 × 104 to 1.24 × 105–1.39 × 105 gene copies g−1, respectively).
Furthermore, the content of tetracycline resistance genes was determined in the DNA samples. About 40 such genes are known from the literature; however, we chose two of them for a number of reasons: firstly, they are responsible for two different mechanisms of antibiotic resistance; secondly, they are among the most frequently analyzed by researchers; thirdly, as a result of our previous research (data not shown), the results obtained for these two genes were the most reproducible [41].
As follows from Figure 3a,b, the patterns of the dynamics of these genes for the control sample were different: at the beginning of composting, the number of copies of the tet(A) gene was 4.89 × 105 copies g−1; by the 7th day it increased to 1.69 × 107 copies g−1, then it decreased, increased, and fell again. The number of copies of the tet(X) gene was higher than that of tet(A) on all days. Moreover, the shape of the curve reflecting the gene copy numbers was different; on the 1st day it showed 1.61 × 107 copies g−1, on the 3rd day it increased to 4.37 × 108 copies × g−1, and then it remained almost constant until the end of the experiment. The addition of biochar, oxytetracycline, and a mixture of metals into compost mixtures did not lead to changes in the nature of the fluctuations in the number of copies of the tet(A) gene. However, it mainly led to their slight increase. Thus, on the 28th day (on which the largest number of gene copies was observed in all samples), the minimum values were noted in samples O, BM, and MO; in other samples, the values over the control were exceeded by 1.6 (sample BO), 2.7 (sample BMO), 2.3 (sample B), and 7.3 (sample M)-fold. At the end of the experiment, the lowest number of copies of the tet(A) gene was also found in the control sample. These differences did not allow us to unambiguously identify the factors influencing the number of copies of antibiotic-resistant genes; therefore, the data were subjected to further statistical processing. In the O, BO, M, and MO samples, the dynamics of the tet(X) resistance genes was similar to that in the control; on the 3rd day, the numbers of gene copies increased by 5–23-fold relative to the control and then remained unchanged until the end of the experiment. The “behavior” of the resistance genes tet(X) in samples B, BM, and BMO is noteworthy; on the 7th day, a significant increase in the number of their gene copies was observed, coinciding with the thermophilic phase. It can be assumed that the carriers of the tet(X) gene in these samples and at that time were thermophilic microorganisms, the number of which sharply decreased after the end of the thermophilic phase. Generally, for both tet(A) and tet(X) genes in all variants of compost mixtures, an increase in the number of gene copies was observed from the beginning of composting to its end.
Integrons of class 1 are mobile genetic elements involved in the horizontal transfer of ARGs. Therefore, assessments of the genes encoding these integrons are widely used to estimate the intensity of the process of horizontal transfer [42]. In the control sample, the number of int1 gene copies on the 1st day of the experiment was 7.26 × 106 copies g−1; on the 14th day it slightly decreased (to 2.3 × 106 copies g−1), and then did not change until the end of the experiment (Figure 3c). A similar pattern (maximum for the 1st day, then a decrease) was observed in all variants of compost mixtures. The highest number of copies of the int1 genes was detected on the 1st day in variants M, MO, and BMO, and on the 14th, 28th, and 84th days in samples O, BO, and MO. It should be noted that in the control variant and the variant with the addition of biochar, the int1 gene was present in slightly lower amounts compared to other composting variants. Additionally, in the sample with biochar we observed the lowest content of int1, and in the sample with heavy metals we observed a maximum drop in the content of int1. This suggests that the addition of antibiotics and metals intensifies the horizontal gene transfer, while the addition of biochar does not affect the transfer. This assumption needs to be verified further using statistics and modeling.

3.3. Bacterial Community Composition of the Composting Mixtures

Since bacteria are carriers of antibiotic-resistant genes, and the present study aims at studying the influence of various factors on the spread of such genes. In addition, it is known from the scientific literature that the fungal community during composting is quite stable and is less affected by factors such as the presence of antibiotics or the introduction of heavy metals, and the analysis of the structure of the bacterial community alone seems to be sufficient to achieve our goals [43].
In total, 283 bacterial OTUs were detected in the studied samples. There were no significant differences in the numbers of OTUs between the variants. On the 1st day, the numbers of OTU in the samples varied from 128 to 170, and by the 84th day, a gradual increase in their number by an average of 1.5–2-fold was observed, which amounted to 255–283 OTUs.
It was found that representatives of the Firmicutes and Actinobacteria phyla were the most abundant at the initial stages of composting. Starting from the 14th day, they were replaced by representatives of the Proteobacteria and Bacteroidetes phyla (Figure 4).
Table 1 presents the OTUs, the abundance of which exceeded 5% on at least one of the sampling days. The similarity in the communities of different composting options turned out to be high. Thus, on the 1st–3rd days of composting, one and the same OTU—Corynebacterium sp.—dominated in all variants (abundance: 14–23%); on the 7th day its abundance decreased to 6–8%, and by the 84th day it was 0–2% in all composting variants. A similar trend towards a decrease in abundance in all variants was demonstrated by Staphylococcus sciuri; on the 1st–3rd days, its abundance was 6–12%, and on the 7th day the range was 3–4%. The peak abundance for species representing the families Bacillaceae and Aerococcaceae was observed on the 3rd day of composting (9–18% and 7–11%, respectively), probably due to their resistance to high temperatures and their ability to decompose organic compounds with heat release. For the OTU of the Sphingobacteriaceae family, an increase in abundance was noted from the beginning of composting (0% in all variants) to the end (4–6%). In general, representatives of the families Corynebacteriaceae, Bacillaceae, Lactobacillales, Planococcaceae, and Staphylococcaceae, found in both the control compost and in composts with various combinations of additives (biochar, antibiotic, and heavy metals), were maximally represented in the thermophilic phase (1–7 days), gradually decreasing in abundance up to the 84th day. Up to the 7th day, bacteria of the families Flavobacteriaceae, Alcaligenaceae, Sphingobacteriaceae, Xanthomonadaceae, and Chitinophagaceae were present in the samples in minimal amounts, but by the end of composting a gradual increase in their abundance was observed.
In general, no significant differences in the dynamics of the main bacteria’s abundance were revealed between the variants (Figure 5). However, some differences were observed between the variants for other OTUs. Thus, on the 28th day, the abundance of Aequorivita sp. was lower in all variants containing heavy metals (M, BM, MO, and BMO) than in other variants. It can be suggested that heavy metals inhibited this species. On the 84th day of composting, differences were noted in the abundance of Gelidibacter sp. in the pairs O–BO and M–BM. Thus, in composts containing OTC and heavy metals (samples O and M), the abundance of the species Gelidibacter sp. turned out to be lower (3%) than in the corresponding composts with the addition of biochar (9%). This was probably due to the mitigating effect of the biochar, which may have partly absorbed the metals.
Similarities and differences between the bacterial communities were assessed using the nonmetric multidimensional scaling (NMDS) method. Figure 6 demonstrates that in general, the bacterial communities of composts containing various combinations of biochar, antibiotic, and heavy metal additives had a high similarity to each other, since the points on the NMDS plot, which represent samples, are located close to each other. The main factor distinguishing bacterial communities from each other was the duration of composting—the points characterizing the samples taken on the same day but for different variants are located close to each other, but far from the points characterizing other days of composting. Interestingly, the groups of points characterizing close sampling days (1–3, 3–7, and 56–84) are located closer to each other than the groups of points characterized by a significant difference in sampling time (for example, 1 and 84 days). Moreover, it can be seen that the similarity in the bacterial communities of different composting variants decreased over time, and the highest similarity was observed during the thermophilic phase of composting (days 3 and 7).

3.4. The Factors Influencing Bacterial Communities and ARG Copy Numbers in the Composting Mixtures

A robust linear model was used to analyze the significance of the effect of the biochar, oxytetracycline, metals, and their combinations on the number of copies of the tet(A), tet(X), and int1 genes. The influence was compared with the control to estimate not only the presence or absence of the influence but also its strength. It was found that the introduction of additives was significant only for the tet(A) gene, but not for the tet(X) gene (Table 2). For the tet(A) gene, the introduction of biochar led to a decrease in the number of copies of genes relative to the control by 26.7%; the introduction of metals led to a decrease of 34.5%, while the introduction of oxytetracycline led to an increase of 43.7%. The number of copies of the int1 gene was significantly affected only by the addition of oxytetracycline, which led to a 45.9% increase.
A correlation between the numbers of tet(A), tet(X), and int1 gene copies and the OTU abundance was carried out using the random forests method. The results are presented in Supplementary Table S1. The abundance of OTUs belonging to the Natronobacillus, Ammoniphilus, Pseudomonadaceae, and Luteimonas taxa was associated with the number of copies of the tet(A) resistance genes, and the abundance of Natronobacillus, Ammoniphilus, Pseudomonadaceae, Luteimonas, and Trichococcus was associated with the number of copies of the tet(X) resistance genes. It can be assumed that these OTUs were carriers of those resistance genes. Interestingly, within the same family and genus, there were OTUs with a high correlation with resistance genes and OTUs with a low correlation. Some examples of such families are Bacillaceae, Planococcaceae, Pseudomonadaceae, and Xanthomonadaceae. Furthermore, for each OTU, the influence (significant or not) of the introduction of the biochar was evaluated via robust linear modeling; in addition, this influence was quantified as a percentage of the control. The OTUs negatively affected by the biochar included representatives of the Dietzia, Gordonia, Kocuria, Natronobacillu, Sporosarcina, Macrococcus, Carnobacteriaceae, Trichococcus, Clostridiaceae, Lachnospiraceae, Coprococcus, Ruminococcaceae, Tissierellaceae, Sporanaerobacter, Tissierella Soehngenia, Alcaligenes faeconaddomaceae, and Luteimonas taxa. Furthermore, we compared the data obtained in two ways and identified those OTUs in which the number of resistance genes was associated with abundance (a coefficient above 70); the effect of the biochar was significant and negative (Table 3 and Supplementary Table S1). These OTUs included representatives of the genus Natronobacillus (association with tet(A) 79%, with tet(X) 86%; influence of biochar 37%), Pseudomonadaceae (association with tet(A) 75%, with tet(X) 80%; influence of biochar 46%), Luteimonas (association with tet(A) 100%, with tet(X) 94%; influence of biochar 34%), and Trichococcus (association with tet(A) 58%, with tet(X) 74%; biochar influence 44%). There were also OTUs with a significant increase in abundance after the addition of the biochar. These were from Corynebacterium variabile and representatives of the Flavobacteriales, Rhodothermaceae, Clostridia, GMD14H09, Halorhodospira, and Piscirickettsiaceae taxa (with increases of 334%, 231%, 292%, 222%, 423%, 1017%, and 287%, respectively). For all these OTUs, no association with the number of ARGs was found. This supports our hypothesis that the biochar creates favorable conditions for the development of species that are not carriers of antibiotic-resistant genes, thereby indirectly influencing the number of carrier species, and accordingly the number of resistance gene copies.

4. Discussion

4.1. Dynamics of the Main Parameters of Composting

The issue of animal waste composting remains relevant and requires further research, since new sequencing methods may reveal the specific characteristics of the composting process, such as the fate and spread of ARGs under the influence of different compost additives. In the present study, it was revealed that neither the biochar nor antibiotic and heavy metals added into compost mixtures have a significant effect on the main physicochemical parameters of composting; in addition, they affect the level of respiratory activity to some extent. The thermophilic phase in all variants of composts was noted on the 2nd–5th days of composting. In general, the obtained temperature profile corresponded to the traditional temperature dynamics during composting described in the literature [44,45,46]. The peak content of dissolved organic carbon was noted on the 14th–21st days for all composts and amounted to 21–32 mg g−1; this result is also in line with what is presented in the scientific literature [47,48,49]. In the final composts, the content of dissolved organic carbon was 3.3–5.3 mg g−1, which corresponds to a high degree of content when the compost is applied to the soil, which contributes to an increase in yield [48,49]. The highest respiration activity was noted on the 3rd day of composting and coincided with the thermophilic stage, which is characterized by a high activity of the microbial community [50,51].

4.2. Dynamics of Copy Numbers of Genes Characterizing Common Bacterial and Fungal Counts, Tetracycline Resistance, and Horizontal Gene Transfer

The number of bacterial gene copies generally corresponds to the scientific literature, as well as the downward trend in this amount during the composting process [52,53,54]. The absence of a decrease in fungal copies of genes revealed in our study was also confirmed by data from other studies [52,55].
For both the tet(A) and tet(X) resistance genes, the lowest copy numbers were found in the control sample. The numbers of both gene copies exceeded the control in samples B, O, BO, M, and MO and were similar to the control values in the other samples. However, the dynamics of the changes in the numbers of copies of these genes differed significantly in the control and experimental variants. Presumably, this was due to the confinement of these genes to one or another group of bacteria that inhabited the composts and changed their counts depending on the temperature, pH, the presence of substrates, and other environmental conditions [15,56,57]. In general, the numbers of copies of the tetracycline resistance genes tet(A) and tet(X) increased from the beginning to the end of the composting by 2–102- and 4–35-fold, respectively. This may have been due to the higher survival rate of the initially resistant species in an oxytetracycline-polluted environment, as well as the transfer of genes to resistant species [58,59]. We found a decrease in the number of copies of the int1 gene encoding a class 1 integron involved in the horizontal transfer of ARGs on the 14th, 28th, and 84th days of composting compared to the 1st day in all variants of compost mixtures. The scientific literature provides contradictory information about the dynamics of the number of copies of the int1 genes during composting. Thus, in a number of works, an increase was noted. Other authors pointed to a downward trend [60,61].
It has been reported that the presence of heavy metals can increase the number of ARGs through co-selection mechanisms (co-resistance, cross-resistance, and co-regulation) [62,63,64,65]. It is also known that the addition of biochar to a compost mixture facilitates the decomposition of antibiotic residues, increasing the surface area for functional groups of bacteria. This results in a change in the number of ARGs in the compost mixtures. By reducing the number of ARGs, the biochar not only reduces its own problem, but also reduces indirect problems (various changes in the composition of bacterial community species) that can be caused by the presence of ARGs [66,67,68]. The addition of antibiotics to a compost mixture can exert selective pressure on the compost bacteria, thereby increasing the resistance of some bacteria and pathogens [69,70,71].

4.3. Composition of Bacterial Communities in the Composting Mixtures

At the phylum level, representatives of Firmicutes and Actinobacteria dominated at the beginning of composting, followed by representatives of Proteobacteria and Bacteroidetes. In general, such a change in the dominant types is typical for mixtures containing straw, sawdust, and chicken manure [72]. Firmicutes are known to produce various proteases and pectinases and can decompose complex carbohydrates such as cellulose. This type of bacteria may play a leading role in nitrogen-rich chicken manure composts [73]. Bacteria belonging to the Actinobacteria play a leading role in the degradation of organic matter and the humification of the composted substrate [74,75]. Proteobacteria and Bacteroidetes take part in the decomposition of complex organic protein compounds, as well as cellulose, starch, and chitin [76,77,78].
In our study, an increase in the number of bacterial OTUs in the process of composting by 1.5–2-fold was observed. The increase in bacterial OTU can be associated with the appearance in the composted substrate of available nutrients for various groups of microorganisms, such as soluble sugars and organic acids [47]. Representatives of the Corynebacteriaceae, Bacillaceae, Lactobacillales, Planococcaceae, and Staphylococcaceae families were most abundant at the beginning of composting, including in the thermophilic phase, in all variants of mixtures. These families are traditionally found in the microbial community of composts and play an active part in the composting process. Thus, bacteria of the Lactobacillales family enhance the degradation of organic substances, such as lignin and cellulose, and exhibit an antagonistic effect against various pathogens in manure by lowering the pH and producing peptides with antimicrobial activity [78]. Representatives of the Flavobacteriaceae, Alcaligenaceae, Sphingobacteriaceae, Xanthomonadaceae, and Chitinophagaceae families dominated at subsequent stages of composting. Bacteria of the Xanthomonadaceae family take part in the decomposition of cellulose. Bacteria of the Alcaligenaceae family are responsible for the degradation of organic acids and amino acids in composts. Bacteria of the Chitinophagaceae family produce enzymes to break down cellulose and chitin [73,78].
When comparing compost mixtures, a high similarity was found at the level of dominant OTUs. At the initial stage of composting (1–3 days), Corynebacterium sp. and Staphylococcus sciuri were characterized by having the highest abundance in all samples (abundance levels of 14–23% and 6–12%, respectively). Further, towards the end of composting, their abundance levels gradually decreased to 0–2% and 0–1%, respectively. Species representing the families Bacillaceae and Aerococcaceae had the maximum abundance levels during the thermophilic phase of composting in all variants (9–18% and 7–11%, respectively), since these species belong to the thermophilic group of microorganisms [75]. Representatives of the families Corynebacteriaceae, Bacillaceae, Lactobacillales, Planococcaceae, and Staphylococcaceae, found in both the control compost and in composts with various combinations of additives (biochar, antibiotic, heavy metals), were highly represented in the thermophilic phase (1–7 days), gradually decreasing in abundance by the 84th day. Up to day 7, the bacteria of the families Flavobacteriaceae, Alcaligenaceae, Sphingobacteriaceae, Xanthomonadaceae, and Chitinophagaceae were present in the samples in minimal amounts, but by the end of composting a gradual increase in their numbers was observed. The various additions to the composts did not have a significant effect on the dynamics of the number of dominant bacterial taxa in the compost mixtures. However, on the 84th day, an increase in the abundance of Gelidibacter sp. in O and M samples containing OTC and metals, compared with BO and BM samples, was registered. This difference can be explained by the fact that the biochar, having a highly porous structure, contributed to the sorption of heavy metals and a decrease in their bioavailability, which affected the abundance of Gelidibacter sp. [79]. Indeed, the literature describes such changes in the structure and dynamics of the microbial community of composts pretreated with biochar [80,81].
The analysis of the beta diversity using the method of nonmetric multidimensional scaling (NMDS) made it possible to establish the high similarity between the bacterial communities in composts containing various additives. However, it was found that the differences between the bacterial communities of the same samples are significantly affected by the duration of composting. Indeed, in the process of composting, there is a natural shift in some bacterial communities by others, depending on the physicochemical characteristics of the composts and the availability of the nutrient substrates [82,83,84,85]. The most similar bacterial communities were observed on the 3rd and 7th days, belonging to the thermophilic phase of composting. This indicates that temperature is one of the main factors that shapes bacterial communities.

4.4. Revealing the Role of Biochar for ARG Fate in the Composting Mixtures

It is known that the use of highly porous substrates can significantly reduce the number of ARGs in the process of manure composting [31,86,87]. For this purpose, biochar is widely used in agriculture, being obtained from such plant materials as rice husks, straw, nut shells, bamboo, and mushrooms. [88,89]. The mechanism of action is mainly due to the high sorption capacity of the biochar in relation to the residual amounts of antibiotics in the composts and a decrease in their bioavailability [90,91,92]. In the present study, another biochar made of chicken manure was used. Using modeling, a significant effect of the introduction of this type of biochar on the number of copies of the tet(A) gene was revealed, whereby the latter decreased by 27% on average. The number of copies of the tet(X) gene was not significantly affected by the addition of biochar. The introduction of oxytetracycline also significantly affected only one of the resistance genes; the number of copies of the tet(A) gene increased by an average of 44%, as well as on the int1 gene, while the number of its gene copies increased by 46%. Interestingly, the addition of biochar, metals, and antibiotics had a much smaller impact on the species diversity at the level of large taxa (phyla, class, and order), but in some cases affected individual OTUs (Pseudomonadaceae, Luteimonas, and Trichococcus). It should be underlined that such a correlation was found specifically for individual OTUs but not for genera or families. Previously, authors have revealed ARG carriers at the genera [25,93,94] or family level [82,83,85].

5. Conclusions

It can be concluded that neither composting nor the addition of chicken manure biochar led to the complete elimination of oxytetracycline resistance genes from chicken manure. However, the addition of biochar caused a significant decrease in the number of ARG copies, including in conditions of contamination with metals and oxytetracycline; therefore, it can be recommended as a measure for the treatment of manure to solve the problem of ARG dissemination in soils and crops.
It was found that bacterial communities of the composted mixtures are very stable and do not depend on the addition of biochar, oxytetracycline, or metals, or on the time of sampling or the characteristics of each individual sample. The main aspect determining the community structure is the composting phase, which is the result of biochemical and physicochemical processes.
It has been shown that the number of copies of ARG in the bacterial community positively correlates with the abundance of several bacterial taxa considered as carriers of ARG. Moreover, it was found that the addition of biochar had a positive effect on some taxa of bacteria (at the OTU level) and a negative effect on several others. Therefore, it has been suggested that the decrease in ARG copy numbers observed in the presence of biochar is due to the creation of more favorable conditions for those species that outcompete ARG carriers from the community.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agriculture12081158/s1, Figure S1: Rotary drum composters used for chicken manure composting; Table S1: Correlation between tet(A) and tet(X) gene copy numbers and OTU abundance and the significance of biochar addition to the composting mixtures.

Author Contributions

Conceptualization, S.S. and P.G.; methodology, P.G.; software, K.K.; validation, K.K., N.D., and P.G.; formal analysis, S.S.; investigation, K.K.; resources, N.D.; data curation, K.K.; writing—original draft preparation, K.K.; writing—review and editing, P.G.; visualization, S.S.; supervision, P.G.; project administration, N.D.; funding acquisition, P.G. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by a subsidy allocated to Kazan Federal University from the Russian Foundation for Basic Research, project No18-29-25054.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

The work is carried out in accordance with the Strategic Academic Leadership Program “Priority 2030” of the Kazan Federal University of the Government of the Russian Federation.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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Agriculture 12 01158 g001a 550Agriculture 12 01158 g001b 550
Figure 1. Temperature (a), dissolved organic carbon content (b), and respiration activity (c) of the compost mixtures.
Figure 1. Temperature (a), dissolved organic carbon content (b), and respiration activity (c) of the compost mixtures.
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Agriculture 12 01158 g002 550
Figure 2. Bacterial (a) and fungal (b) gene copy numbers in the compost mixtures.
Figure 2. Bacterial (a) and fungal (b) gene copy numbers in the compost mixtures.
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Agriculture 12 01158 g003a 550Agriculture 12 01158 g003b 550
Figure 3. Number of copies of the tet(A) gene (a), tet(X) gene (b), and the gene encoding integron class A (int1) (c) in the composting mixtures.
Figure 3. Number of copies of the tet(A) gene (a), tet(X) gene (b), and the gene encoding integron class A (int1) (c) in the composting mixtures.
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Agriculture 12 01158 g004 550
Figure 4. Bacterial community structure of the composting mixtures (phylum level).
Figure 4. Bacterial community structure of the composting mixtures (phylum level).
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Agriculture 12 01158 g005 550
Figure 5. The dynamics of dominant OTUs in the compost mixtures during the composting process.
Figure 5. The dynamics of dominant OTUs in the compost mixtures during the composting process.
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Agriculture 12 01158 g006 550
Figure 6. Beta-diversity of the bacterial communities of the compost mixtures (numbers represent the days of sampling and letters the names of the variants).
Figure 6. Beta-diversity of the bacterial communities of the compost mixtures (numbers represent the days of sampling and letters the names of the variants).
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Table
Table 1. The list of dominating OTUs in the compost mixtures, and their abundance levels during the composting process.
Table 1. The list of dominating OTUs in the compost mixtures, and their abundance levels during the composting process.
NoPhylumClassOrderFamilyGenusSpecies
1ActinobacteriaActinobacteriaActinomycetalesCorynebacteriaceaeCorynebacterium
2FirmicutesBacilliBacillalesBacillaceae
3BacteriaFirmicutesBacillLactobacillalesAerococcaceae
4BacteroidetesFlavobacteriiaFlavobacterialesFlavobacteriaceae
5ProteobacteriaBetaproteobacteriaBurkholderialesAlcaligenaceae
6FirmicutesBacilliBacillalesStaphylococcaceaeStaphylococcusStaphylococcus sciuri
7BacteroidetesSphingobacteriiaSphingobacterialesSphingobacteriaceae
8FirmicutesBacilliBacillalesStaphylococcaceaeStaphylococcus;Other
9OtherOtherOtherOtherOtherOther
10FirmicutesBacilliBacillalesStaphylococcaceaeJeotgalicoccus
11FirmicutesBacilliBacillalesPlanococcaceae
12BacteroidetesFlavobacteriiaFlavobacterialesFlavobacteriaceaeAequorivita
13ActinobacteriaActinobacteriaActinomycetalesYaniellaceaeYaniella
14FirmicutesBacilliLactobacillalesLeuconostocaceaeWeissellaOther
15FirmicutesBacilliLactobacillalesLactobacillaceaeLactobacillus
16ActinobacteriaActinobacteriaActinomycetalesDermabacteraceaeBrachybacteriumBrachybacterium conglomeratum
17BacteroidetesFlavobacteriiaFlavobacterialesFlavobacteriaceaeGelidibacter
18ActinobacteriaActinobacteriaActinomycetalesBrevibacteriaceaeBrevibacterium
19FirmicutesBacilliBacillalesStaphylococcaceaeStaphylococcus
20ActinobacteriaActinobacteriaActinomycetalesOtherOtherOther
21ProteobacteriaGammaproteobacteriaAlteromonadalesAlteromonadaceae
22BacteroidetesFlavobacteriiaFlavobacteriales[Weeksellaceae]
23FirmicutesBacilliBacillalesBacillaceaeOtherOther
24[Thermi]DeinococciDeinococcalesTrueperaceaeB-42
25ProteobacteriaGammaproteobacteriaPseudomonadalesPseudomonadaceaePseudomonas
26BacteroidetesCytophagiaCytophagalesFlammeovirgaceae
27ProteobacteriaGammaproteobacteriaXanthomonadalesXanthomonadaceaeLuteimonas
28ProteobacteriaGammaproteobacteriaPseudomonadalesPseudomonadaceae
29ProteobacteriaGammaproteobacteriaXanthomonadalesXanthomonadaceaeRhodanobacter
30Bacteroidetes[Saprospirae][Saprospirales]Chitinophagaceae
Table
Table 2. The influence of the additives (biochar, oxytetracycline, metals) on the number of copies of the tet(A), tet(X), and int1 genes.
Table 2. The influence of the additives (biochar, oxytetracycline, metals) on the number of copies of the tet(A), tet(X), and int1 genes.
Subject of InfluenceInfluencing FactorMean Changes in Values Relative to Control (V)p-ValueChanges Expressed in % Relative to Control (y = 100 × (exp(−V) − 1))
tet(A)Biochar addition−0.310130.0246−26.66491924
Oxytetracycline addition0.3624130.008643.67922146
Metals addition−0.422410.0022−34.45332489
tet(X)Biochar addition−0.055320.2467-
Oxytetracycline addition0.0887840.0632-
Metals addition−0.049610.2989-
int1Biochar addition−0.06371190.5848-
Oxytetracycline addition0.377762080.001245.90157725
Metals addition0.14560250.2117-
Table
Table 3. Correlation between tet(A) and tet(X) gene copy numbers and OTU abundance levels and the significance of the biochar addition to the composting mixtures (fragment). The entire table is presented in Supplementary Table S1.
Table 3. Correlation between tet(A) and tet(X) gene copy numbers and OTU abundance levels and the significance of the biochar addition to the composting mixtures (fragment). The entire table is presented in Supplementary Table S1.
OTECorrelation between tet(A) Gene Copy Number and OTU Abundance, % from MaximumCorrelation between tet(X) Gene Copy Number and OTU Abundance, % from MaximumSignificance of Biochar Addition to the Composting Mixtures
p-ValueMean Changes as Compared with Control (V)Changes in % as Compared with Control y = 100 × (exp(−V) − 1)Average Abundance, %
Natronobacillus sp.79860.013−0.4564−36.64
Ammoniphilus sp.75790.01730.493263.81
Pseudomonadaceae; g__; s__75800.032−0.6208−46.28
Luteimonas; s__100940.0314−0.4157−34.08
Trichococcus; s__58740.0033−0.5886−44.51
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Karamova, K.; Danilova, N.; Selivanovskaya, S.; Galitskaya, P. The Impact of Chicken Manure Biochar on Antibiotic Resistance Genes in Chicken Manure Composting. Agriculture 2022, 12, 1158. https://doi.org/10.3390/agriculture12081158

AMA Style

Karamova K, Danilova N, Selivanovskaya S, Galitskaya P. The Impact of Chicken Manure Biochar on Antibiotic Resistance Genes in Chicken Manure Composting. Agriculture. 2022; 12(8):1158. https://doi.org/10.3390/agriculture12081158

Chicago/Turabian Style

Karamova, Kamalya, Natalia Danilova, Svetlana Selivanovskaya, and Polina Galitskaya. 2022. "The Impact of Chicken Manure Biochar on Antibiotic Resistance Genes in Chicken Manure Composting" Agriculture 12, no. 8: 1158. https://doi.org/10.3390/agriculture12081158

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