Effects of nanoparticles on the anaerobic digestion properties of sulfamethoxazole-containing chicken manure and analysis of bio-enzymes

2.3.1 Reagents

Preparation of the nano-Fe₂O₃ suspension: nano-Fe₂O₃ (90 nm) was purchased from Shanghai Macklin Biochemical Co., Ltd., with a purity of 99.8%.

Preparation of the nano-C₆₀ suspension: nano-Fe₆₀O₃ (90 nm) was purchased from Shanghai Macklin Biochemical Co., Ltd., with a purity of 99.9%.

SMX (98%, CAS:723-46-6) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., with a chemical formula of C₁₀H₁₁N₃O₃S, a relative molecular weight of 253.28, and a purity of ≥98%. SMX was preserved at 4°C in the fridge before use.

2.3.2 Experiments

Six groups were set up, and each group was added with 120 g of chicken manure and 300 g of bacteria-inoculated slurry. The maximum residue concentration of SMX was 18.00 mg·kg⁻¹·TS and was rounded up to 20 mg·kg⁻¹·TS. SMX was added in one dose at 50, 100, and 200% of the maximum residue concentration, respectively. In groups S4 and S5, under the SMX concentration of 20 mg·kg⁻¹·TS, 300 mg·kg⁻¹·TS nano-Fe₂O₃ and 100 mg·kg⁻¹·TS nano-C₆₀ were added, respectively. The amounts of each ingredient added are listed in Table 2. After feeding, deionized water was added to reach a final volume of 1 L, followed by nitrogen stripping for 2 min to remove the air. The tank was then sealed. The anaerobic digestion reactor was placed in a thermostatic water bath at 37 ± 0.5°C and shielded from light. Four replicates were set up for each group. One replicate was only used for supplementing the material lost for the sampling detection, and the final result of each group was the average of the remaining three replicates. Throughout the experiments, the slurry was stirred manually twice daily, for 1 min each time.

2.4.1 Analysis of physicochemical properties

The conventional indicators determined in the experiments were mainly related to the supernatant of the digestive fluid, including volatile fatty acids (VFAs) and pH value. The slurry supernatant was obtained by centrifugation of an appropriate amount of the digestion mixture using a high-speed refrigerated centrifuge at 9,000 rpm for 10 min, followed by filtration using a 0.45 μm filter membrane [6].

The physiochemical properties were detected as follows:

1. Volatile fatty acids: The composition and content of VFAs were determined using a gas chromatogram. The slurry supernatant was then passed through a 0.22 μm filter membrane. Into a sample vial, 1 ml of the sample was added, along with 100 μL of chromatographic grade formic grade to acidify the sample to pH < 2.

   Biogas production was measured by the water displacement method once daily.

   The VFA concentration was determined by gas chromatography under the following conditions: chromatographic column: stainless steel packed column manufactured by National Chromatographic R&A Center, Dalian Institute of Chemical Physics, with TDX-01 as filler measuring 2 m × 3 mn; detector: thermal conductivity detector; carrier gas: He, flow rate 20 mL·min⁻¹; current: 100 mA; attenuation: 1; detection temperature: 200°C; column box temperature: 180°C; injection temperature: 200°C.

2. pH value: The pH value was determined using the glass electrode method.

2.4.2 Determination of SMX content

5 g of the sample was precisely weighed and added with 20 mL of acetonitrile, and homogenized, vortexed, and ultrasonically treated for 10 min, respectively. Next, 5 g of anhydrous sodium sulfate was added, vortexed (without causing agglomeration), and centrifuged at 10,000 rpm for 10 min. The supernatant was transferred to a chicken heart bottle and added with 7 ml of isopropyl alcohol. After ultrasonic treatment for 1 min, the mixture was concentrated in a rotary evaporator at 45°C until reaching a volume of about 5 mL. The above process was repeated. The chicken heart bottle was washed with a total of 15 ml of 0.1 mol·L⁻¹ HCl three times, and the fluid was collected into a 50 mL centrifuge tube after washing. Next, the chicken heart bottle was washed with 5 mL of n-hexane, vortexed for 2 min, and centrifuged at 10,000 rpm for 5 min. The lower n-hexane layer was discarded, and the remaining portion was used for purification.

Solid phase extraction was performed using an MCX column (60 mg, 3 mL), which was activated using 3 mL of methanol and 3 ml of 0.1 mol·L⁻¹ HCl successively. Then, 8 ml of the analyte was precisely weighed and loaded. After the elution with 3 mL of 0.1 mol·L⁻¹ HCl and 3 mL of 50% methanol in aqueous solution and draining of the waste liquid, elution was performed again using 5 mL of 5% methanol in aqueous ammonia. Nitrogen stripping was performed at 50°C, and the analyte was redissolved using 0.2% formic acid in aqueous solution: methanol (1:1). After passing through a 0.22 μm organic filter membrane, the analyte was bottled and loaded for detection.

2.4.3 Determination of enzyme activity

According to the manufacturer’s instructions, the corresponding enzyme kit (Shanghai Enzyme-Linked Biotechnology Co., Ltd., China) was used to determine the activities of amylase, cellulase, protease, urease and dehydrogenase. The procedures for different types of enzymes are the same as follows:

An appropriate amount of anaerobic digestion liquid was broken using an ultrasonic cell crusher (750 W, 40% power, 5 s ultrasound, 9 s interval) in an ice bath for 90 min, and the broken sample was centrifuged at a low temperature (4°C) at a rotational speed of 10,000 rpm for 20 min. The supernatant was taken and stored at −20°C for measurement. Standard product holes were set, and standard product holes were added at different concentrations (400, 200, 100, 50, 25, 0 IU·L⁻¹) 50 μL of enzyme-labeled reagent; A blank control hole and a sample hole to be measured were set up respectively. The sample and enzyme-labeled reagent are not added to the blank control hole, and the other steps are the same. The sample diluent 40 μL is added to the sample hole to be measured first, and then 10 μL of the sample to be measured is added, that is, the final dilution of the sample is 5 times. 100 μL of enzyme-labeled reagent was added to each well, the plate was sealed with the sealing plate film and incubated at 37°C for 60 min. The sealing plate film was removed, the liquid was discarded, and shaken dry, each hole was filled with 20 times diluted washing liquid, left for 30 s and discarded, repeated 5 times and pat dried. 50 μL of color developer A was added to each well first, then 50 μL of color developer B was added to each well, shocked and mixed, color development was carried out at 37°C in the dark for 15 minutes, and 50 μL of termination solution was added to terminate the reaction, at this time, the blue turned to yellow immediately, and the determination was performed within 15 min. The absorbance of each hole was measured at 450 nm wavelength and the standard curve was drawn.

2.4.4 Metagenomic sequencing

Appropriate amounts of chicken manure and inoculated sludge were taken and stored at −20°C to be measured. Metagenomic sequencing was completed by Shanghai Meiji Biomedical Technology Co., Ltd. The samples were extracted using a FastDNA^® Spin Kit for Soil extraction kit according to the instructions. Illumina’s NovaSeq sequencing platform was used for metagenomic sequencing. After the optimized sequences were spliced and assembled, the high-quality reads of each sample were compared with the non-redundant gene set (95% identity), and the abundance information of the genes in the corresponding sample was calculated. The amino acid sequence of the non-redundant gene set was compared with the CARD database to obtain the annotation information of the antibiotic resistance function corresponding to the gene, and then, the abundance of the antibiotic resistance function was calculated using the sum of the gene abundance corresponding to the antibiotic resistance function.

3.1.1 Effects of nanoparticles on the anaerobic biogas production from SMX-containing chicken manure

Changes in daily biogas production and cumulative biogas production from the anaerobic digestion of SMX-containing chicken manure in the presence of nanoparticles are shown in Figure 2. In all six groups, two peaks of biogas production occurred during 55 days of anaerobic digestion. The first peak lasted from day 1 to day 10 of the experiment. Once the experiment was started, hydrolytic acid-producing bacteria broke down the dissolved organic matter at a fast rate. At this stage, there was an adequate nutrient supply for anaerobic digestion by methanogens, which was coupled with an appropriate pH value to sustain a high methanation efficiency. In S1–S3 without the addition of nanoparticles, the peak was reached on day 1, the biogas production being 443 ± 23, 264 ± 19, and 270 ± 19 mL, respectively. The first peak lagged behind in the blank control group CK compared with the other groups and occurred on day 5, the value being 340 ± 18 mL. The peak occurred as early as day 1 in S4 added with nanoparticles, the biogas production reaching 345 ± 22 mL. The first peak also occurred on day 1 in S5 added with nanoparticles, reaching 472 ± 22 mL. The cumulative biogas productions in the six groups were ranked as follows: 2,570(S5)＞1,861(S4)＞1,420(S1)＞1,375(S2)＞1,372(CK)＞1,114(S3) mL. It can be seen that both nano-Fe₂O₃ and nano-C₆₀ promoted anaerobic digestion to produce biogas from chicken manure containing varying concentrations of SMX. In the first 10 days of the digestion reaction, the cumulative biogas production increased by 35.4% and 130.7% in S4 and S5, respectively. After the first peak, the biogas production reached a stagnation in all groups. NaOH was added from day 21 to day 23 to adjust the pH value to above 6.5. The biogas production was restored to the previous rate in different groups after day 24, and the second peak came soon afterwards. At this stage, insoluble macromolecular organics, including cellulose, were degraded by hydrolytic acid-producing bacteria into VFAs, providing nutrients for methanogens. The biogas production rate increased rapidly in S1 and S2. That is, the second peak of 231 mL was reached on day 34 in S1, and the second peak of 185 mL was reached on day 40 in S2. For S3, the second peak of 390 mL occurred on day 45, and for the CK group, the second peak of 345 mL occurred on day 48. As seen above, compared with S1 and S2, CK and S3 had an extended delay in reaching either peak, though the peaks were larger in amplitude and shorter in duration for CK and S3. A study [7] showed that sulfonamides exert a solubilizing action by damaging extracellular polymeric substance (EPS) and cell membranes while enhancing acid and acetic acid production. This may be one reason for the high biogas production efficiency in S1 and S2. In contrast, due to a higher SMX concentration, bacterial inhibition was observed in S3. Another peak of biogas production of 272 ± 15 ml and 310 ± 8 mL appeared on day 30 and day 33 in S1 and S2, respectively. The peak of 230 ± 11 mL was reached on day 35 in the CK group. In S4 and S5 added with nanoparticles, the second peak was reached on day 39 and day 40, respectively, the value being 216 ± 12 mL and 225 ± 4 mL, respectively. The maximum daily biogas production rate at the second peak was lower in S4 and S5 compared with the other four groups. The final cumulative biogas productions in the six groups were as follows: 4,182(CK), 4,774(S1), 4,459(S2), 4,245(S3), 4,909(S4), and 5,450(S5) mL. As a result, the conversion of carbon dioxide to methane was accelerated, and the delay in the initiation of biogas production was shortened, resulting in higher methane production.

Figure 2

Effects of nanoparticles on changes in daily biogas production rate and cumulative biogas production from anaerobic digestion of SMX-containing chicken manure.

Ma et al. [19] fed sulfadimidine continuously into the anaerobic digestion experiments of cow manure. The results showed that after six days of experiment, the biogas production efficiency increased significantly and the biogas production was finally increased by 44.8%. Their results agreed with ours. That is, the addition of antibiotics did not change the overall biogas production. After acclimation, the biogas production efficiency of the anaerobic digestion system was improved considerably. Wen et al. [20] showed that 50 mg·L⁻¹ SMX increased methane production by high-temperature anaerobic digestion by 22%, probably because SMX acted as a substrate. Currently, there are two explanations for antibiotics promoting biogas production by anaerobic digestion. The first is that antibiotics act as substrates to provide a carbon supply for bacteria [21,22]; the second is that antibiotics have stimulating effects on the bacterial community [23]. Although SMX was almost completely degraded in the earlier stage of the experiment, the theoretical contribution of SMX as a substrate was far lower than the increment of biogas production. This result indicates the positive role of SMX on the bacterial community. Our biogas production data showed that SMX at a residue concentration promoted anaerobic digestion to produce biogas. But as the SMX concentration increased, the biogas production-promoting effect was weakened, while the bacterial inhibitory effect of antibiotics was gradually manifested. It is safe to say that SMX residues in chicken manure were inadequate to cause a negative impact on the anaerobic digestion system.

Adding nano-Fe₂O₃ and nano-C₆₀ nanoparticles had a promoting effect on biogas production by anaerobic digestion of chicken manure containing different concentrations of SMX. The biogas production efficiency increased considerably in the first 10 days, and the cumulative biogas production in the groups added with nanoparticles was higher compared with those not added with nanoparticles. Nanoparticles nano-Fe₂O₃ and nano-C₆₀ deliver extraordinary performance barely comparable with other materials due to their unique structural features. The ultrastructure of the nanoparticles gives rise to surface effects, which manifest as an abrupt increase in nanoparticles’ surface area, surface energy, and surface tension as the diameter decreases. From the above-mentioned reasons, nanoparticles have higher chemical activities compared with other materials and hence a higher possibility of reacting with other chemical substances. Nanoparticles are considered to serve as the mediator promoting gas production by bacteria that hydrolyze the substrates. Another study shows that nanoparticles help increase the metabolic rate and hydrogen yield of substrates involved in anaerobic fermentation. Adding an appropriate concentration of nanoparticles into an anaerobic fermentation hydrogen production system can increase biogas yield by elevating the hydrogenase activity and optimizing the composition of the anaerobic microbial community. The dissolution of metal ions from nano-Fe₂O₃ and nano-C₆₀ is considered one of the most common and also the most important pathways by which nanoparticles affect the anaerobic digestion of slurry. Fe³⁺ is an essential trace element for the anaerobic growth and metabolism and plays a positive role within an appropriate range of concentrations. One study arrived at similar conclusions, believing that at a lower concentration of nano-Fe₂O₃, the released Fe³⁺ was chelated with the negatively charged residues in the EPS, reducing the ionic toxicity.

3.1.2 Effects of nanoparticles on the changes in pH and total volatile fatty acids (TVFA) levels during anaerobic digestion of SMX-containing chicken manure

The effects of nanoparticles on the changes in pH value and TVFA levels during anaerobic digestion of chicken manure containing different concentrations of SMX are shown in Figures 3 and 4, respectively. The changes in the pH value during anaerobic digestion under different SMX concentrations are shown in Figure 3. After the first peak (day 1 to day 5), the anaerobic digestion system in each group began to be acidified. There was a downward trend in the pH value from day 6 to day 20. The pH value of the CK group was the lowest among the four groups, while those of S1 and S2 were close to each other and higher than that of S3. The pH values in S4 added with 300 mg·kg⁻¹·TS nano-Fe₂O₃ and in S5 added with 100 mg·kg⁻¹·TS nano-C₆₀ were even lower. On day 9, the pH values in these two groups were 5.35 ± 0.7 and 5.31 ± 0.6, respectively, indicating that adding nanoparticles contributed significantly to substrate degradation. After the pH adjustment, the pH value decreased in all groups. Later, the pH value first increased on day 30 and day 32 in S1 and S2, respectively. In these two groups, the pH value was restored to above 7 on day 34 and day 38, respectively. From day 34 to day 39, the pH values of CK and S3 were significantly lower than those of the other two groups until an obvious rise in the pH value occurred after day 39. Such changes in pH value were consistent with the variations in biogas production.

Figure 3

Effects of nanoparticles on changes in pH value during anaerobic digestion of the SMX-containing chicken manure.

Figure 4

Effects of nanoparticles on changes in TVFA levels during anaerobic digestion of the SMX-containing chicken manure.

VFAs are the products of hydrolytic acid-producing bacteria as well as the nutrients for methanogens. The VFA levels were dynamically changing in the anaerobic digestion system. The changes in the TVFA levels in the anaerobic digestion system under different SMX concentrations are shown in Figure 4. In different groups, the TVFA levels first increased and then decreased in the earlier stage of the experiment. The TVFA levels were lower in S1 in the earlier stage, indicating a higher substrate utilization rate in the bacterial community in this group. From day 1 to day 10, the TVFA levels in all groups increased rapidly, and it was consistently higher in S4 and S5 compared with the other four groups. It was inferred that the presence of nano-Fe₂O₃ and nano-C₆₀ promoted the hydrolysis of chicken manure containing SMX. The ultrastructure of the nanoparticles facilitated substrate degradation by hydrolytic acidifying bacteria, resulting in a higher TVFA levels in the first 10 days compared with other groups. Later, the pH value decreased, and the cumulative biogas production from TVFAs also decreased. Another peak was reached in S1 and S2 on day 30, the values being 8,104.00 and 7,689.55 mg·L⁻¹, respectively. The corresponding pH values were 6.53 and 6.42, respectively. The biogas production increased continuously in CK and S3 until peaking on day 34, with the values of 10,140.23 and 9,187.95 mg·L⁻¹, respectively. The corresponding pH values were 6.73 and 6.61, respectively. At this stage, biogas production had reached a stagnation point. CK and S3 had not yet recovered from the acid inhibition on methanogens. Studies have shown that the derivatives of some antibiotics still display biotoxicity, and the products of SMX degradation deserve further investigation [24,25]. Latif et al. [26] found that when the pH value was 5.5 in the anaerobic digestion system, the intense accumulation of propionic acid and butyric acid led to a 50% reduction in methane production, while the organic matter not completely degraded accounted for an increasing proportion. When the pH value was below 5.5, the methanogenic community shrank in size considerably and gave way to the propionic acid-utilizing community. In the present study, the pH level was even lower after the addition of nano-Fe₂O₃ and nano-C₆₀, the values being 5.08 ± 0.4 and 5.03 ± 0.2, respectively, which agreed with the findings with TVFAs. Addition of 300 mg·kg⁻¹·TS nano-Fe₂O₃ and 100 mg·kg⁻¹·TS nano-C₆₀ promoted organic acid production from substrate hydrolysis, resulting in a lower pH value of the system. After the addition of NaOH, the pH value of each group was restored to 6.65 to 6.74 on day 26. As biogas production was resumed in each group, the TVFA levels decreased rapidly, and the biogas production rate reached the maximum in each group since day 30. From day 34 to day 37, another rapid increase in TVFAs was observed, indicating that the hydrolysis rate of difficult-to-degrade substances, such as cellulose, was higher than the nutrient utilization rate of methanogens. As the substrate concentration decreased after day 37, the density of hydrolytic acid-producing bacteria decreased as well, accompanied by a gradual reduction in the TVFA concentrations. From day 25 to day 34, the TVFA levels remained low, although the biogas production rate was inconsistent with the low TVFA levels. The low TVFA levels in the system were not due to the utilization of methanogens. Enrofloxacin might have a negative impact on hydrolytic acid-producing bacteria [27]. One study [28] showed that the bacterial inhibition effect of antibiotics reduced methane production without causing acetic acid accumulation, and the working mechanism may be related to the non-competitive inhibition of the enzymes. That is, the enzyme-substrate complexes for antibiotics binding deactivated the enzyme sites, thus inhibiting methane production from acetic acid.

3.1.3 Effects of nanoparticles on the degradation rate of SMX

The SMX degradation rates by anaerobic digestion of chicken manure added with nanoparticles are listed in Table 3. SMX was decomposed rapidly from day 1 to day 5. The SMX degradation rates in the groups added with nano-Fe₂O₃ and nano-C₆₀ were slightly higher than those in the other three groups. On day 5, the SMX degradation rates reached up to 99.64–99.81% in the two groups added with nanoparticles; on day 15, the SMX degradation rates were as high as 99.78–99.94%. In the earliest stage of the experiment, SMX was almost completely removed, indicating the low durability of SMX in the anaerobic digestion system, which agreed with the results of other researchers. Mohring et al. [29] found that SMX was almost completely removed during the 34-day medium-temperature anaerobic digestion experiments in pig manure. Wen et al. [30] estimated the half-life of SMX to be 3 days at a concentration of 50 mg·L⁻¹ during the high-temperature anaerobic digestion experiments in batch reactors. It has been found that in the absence of nanoparticles, absorption is the primary removal pathway for SMX in the anaerobic digestion system [31]. We performed solid phase extraction for the samples to be analyzed in the present study, and the estimated SMX concentration was the total concentration (SMX in the solid and liquid phases combined). The reactors were shielded from light during the experiments, and therefore, the influence of photodegradation was excluded. Medium-temperature anaerobic digestion was effective in removing SMX, according to our results. Addition of 300 mg·kg⁻¹·TS nano-Fe₂O₃ or 100 mg·kg⁻¹·TS nano-C₆₀ further degraded SMX on the basis of the conventional medium-temperature anaerobic digestion. The results suggested that the nanoparticles acted synergistically with SMX in promoting anaerobic metabolism.

3.1.4 Effects of SMX on the activities of different enzymes

Anaerobic digestion to produce biogas is the result of stepwise enzymatic reactions mediated by bacterial communities with different functions. Dehydrogenase activity is an important indicator characterizing microbial state. Changes in dehydrogenase activity during anaerobic digestion under different SMX concentrations are shown in Figure 5. Throughout the experiments, the dehydrogenase activity in the SMX-containing groups was consistently higher than in the control group. The mean dehydrogenase activities in different groups were ranked as follows: 132.44(S5) > 126.52(S4) > 119.38(S2) > 114.88(S3) > 111.57(S1) > 100.78(CK) IU·L⁻¹. Besides, the dehydrogenase activity was the highest by adding 300 mg·kg⁻¹·TS nano-Fe₂O₃ then adding 100 mg·kg⁻¹·TS nano-C. Yang et al. [32] performed a pig manure composting experiment and found that 95 mg·kg⁻¹·TS SMX activated microbial dehydrogenase activity, but inhibited dehydrogenase at a concentration of 50 mg·kg⁻¹·TS. They believed that in an environment with high SMX concentration, the growth and proliferation of resistant bacteria require a large nutrient supply, resulting in an elevated dehydrogenase activity. In contrast, a low SMX concentration in the environment promoted the accumulation of superoxides, which further had a toxic effect on the microbes.

Figure 5

Changes in dehydrogenase activity at different SMX concentrations.

The hydrolysis rate has a limiting effect on the overall anaerobic reaction rate and directly affects the biogas production potential. The hydrolytic state can be intuitively represented by the hydrolase activity. Based on the main components of the substrates, we monitored the activities of four extracellular hydrolases at different states, namely, amylase, cellulase, protease, and urease. The changes in hydrolase activities during anaerobic digestion under different SMX concentrations are shown in Figure 6. Throughout the experiments, the mean amylase activities in different groups were ranked as follows: 400.44(S2) > 368.41(S3) > 349.88(S5) > 347.88(S4) > 329.68(S1) > 281.65(CK) IU·L⁻¹. The mean amylase activities were always higher in the groups added with antibiotics compared with the control group. In contrast, adding nanoparticles barely had any promoting effect. In the earlier stage of the experiment, the amylase activity in S2 on day 1 was significantly higher than that in the other three groups. However, there was a rapid increase on day 5, and the amylase activity in S2 was lower than that in S1 and S3. One reason may be that the antibiotics accelerated amyloysis by hydrolytic bacteria. As the substrate was being rapidly depleted, the bacterial population density decreased, leading to a lower amylase activity.

Figure 6

Changes in activities of hydrolytic enzymes at different SMX concentrations.

Chicken manure is rich in difficult-to-degrade substances, such as coarse fibers, and cellulase activity can reflect the level of cellulose degradation in the anaerobic digestion system. During the experiments, the mean cellulase activities in different groups were ranked as follows: 232.44(S3) > 216.84(S2) > 206.64(S4) > 203.32(S5) > 203.50(S1) > 172.62(CK) IU·mL⁻¹. At different stages of the experiment, the cellulase activities in the groups added with antibiotics were significantly higher than those in the control group. The cellulase activity first increased and then decreased in different groups. As the SMX concentration increased, the cellulase activity increased as well, indicating that SMX simulated the bacterial secretion of cellulase. However, the cellulase concentrations in the groups added with nanoparticles were lower than S2, indicating that nanoparticles only had a limited promoting effect on fibers. On day 15, the maximum cellulase activity reached 199.64, 240.92, 242.39, 280.85, 243.4, and 241.2 IU·mL⁻¹ in the six groups, respectively. The crystalline region of cellulose is difficult to degrade due to its dense structure, and therefore it takes more time to be decomposed into glucose [33]. Cellulose is the main contributing substrate for the second biogas production peak. For this reason, the cellulase activities in the middle and later stages of the experiment were significantly higher than those in the earlier stage. On day 50, the cellulase activities decreased in all groups, and there was a narrowing of intergroup differences, indicating that the hydrolysis of cellulose was completed over time.

Crude protein accounts for a large proportion of the total organic matter in chicken manure. Therefore, protease activity was the key indicator requiring monitoring in our study. Throughout the experiments, the mean protease activities in different groups were ranked as follows: 216.99(S2) > 206.12(S5) > 203.44(S4) > 201.75(S3) > 190.81(S1) > 160.62(CK) IU·L⁻¹. SMX had an obvious promoting effect on protease activity. In the earlier stage of the experiment, the protease activity in S1 was significantly higher than that in S2 and S3. This is probably because SMX inhibited proteolytic bacteria at the concentrations of 20 and 40 mg·kg⁻¹·TS. As the inhibitory effect of SMX on the bacteria was diminished, a highly active state of the bacteria was maintained in S2 and S3 in the middle and later stages of the experiment. This situation was consistent with the variation in the activities of cellulase and amylase. In contrast, the presence of nanoparticles had no obvious impact on protease activity.

Urease converts the C–N bond in amides into ammonia to maintain the nitrogen cycle. In the experiments, the mean urease activities in different groups were ranked as follows: 61.59(S2) > 56.69(S3) > 56.38(S5) > 55.46(S4) > 52.06(S1) > 45.46(CK) IU·mL⁻¹. The urease activity was continuously lower in the control group than in the groups added with antibiotics, which agreed with the results for other extracellular enzymes. SMX did have a stimulating effect on urease activity. Han et al. [34] found that SMX-containing biogas residues dramatically improved the soil urease activity.

3.2.1 Effects of nanoparticles on bacterial communities

Relative abundances of bacteria at the phylum level during anaerobic digestion of chicken manure containing SMX are shown in Figure 7. The bacterial community structures in CK, S2, S4, and S5 were similar at different stages of the experiment on the phylum level. The bacterial community was mainly composed of the following seven phyla: Bacteroidota, Actinobacteriota, Spirochaetota, Proteobacteria, Chloroflexi, and Synergistota. Firmicutes, Bacteroidota, and Proteobacteria are common bacterial communities in anaerobic digestion reactors and can metabolize a variety of substrates. These bacteria are mainly engaged in the hydrolysis of macromolecular substances and organic acid production. Firmicutes can produce large amounts of extracellular enzymes, including protease, cellulase, and lipase [35,36]. Bacteroidota plays an important role in decomposing carbohydrates and proteins into acetic acid and NH₃. Some genera belonging to the Proteobacteria have the capacity of reducing sulfates into H₂S. Spirochaetota, Synergistota, and Chloroflexi can decompose the organic acids produced by hydrolytic acidifying bacteria into acetic acid and H₂ [37]. On day 1, Firmicutes and Bacteroidota were the main dominant bacterial phyla, with relative abundances of 45.13–68.53% and 26.12–48.32%, respectively. Addition of nanoparticles increased the abundances of Bacteroidota in S4 and S5 significantly. On day 5, the relative abundances of Actinobacteriota and Bacteroidota increased dramatically, reaching 6.34–20.28% and 33.11–65.49%, respectively. The abundance of Bacteroidota was slightly higher in the groups added with nanoparticles compared with S2. This is because nanoparticles acted as interfacial mediators to promote the increase in the abundance of Bacteroidota. From day 15 to day 20, Firmicutes gained an increasing competitive advantage as the reaction proceeded, while the relative abundance of Bacteroidota kept decreasing, probably due to a lack of nutrient supply. However, the abundance of Bacteroidota in S4 and S5 added with nanoparticles was higher compared with the other groups. On day 50, Firmicutes became the dominant phylum, and there was a narrowing of intergroup differences in its abundance. The relative abundance of Firmicutes varied between 90.87% and 94.54%.

Figure 7

Effects of nanoparticles on structural changes of the bacterial community involved in the anaerobic digestion of SMX-containing chicken manure on the phylum level.

As shown in Figure 8, at different stages of the experiment, the bacterial community structure at the genus level was dramatically affected by substrates. As nutrients were being depleted, some bacterial communities lost their original competitive advantages. On day 5, the relative abundance of Prevotella belonging to the phylum Bacteroidota increased, indicating a high bacterial resistance to SMX. Especially, the relative abundance of Prevotella in S4 and S5 added with the nanoparticles was lower than that of S2 by 8–10%. On day 15, compared with the control group CK, the relative abundance of Prevotella decreased in S2 as well as in S4 and S5, although the decrease was smaller than in S2. This is probably because the easy-to-degrade nutrients in the substrates had been rapidly decomposed from day 1 to day 10. The hydrolysis acidification process was more intense at the earlier stage in S4 and S5, resulting in a deficiency of nutrients in the substrates, which explains the reduced relative abundance of Prevotella. At this stage, the relative abundances of Treponema were higher in S2, S4, and S5, reaching 4.37% to 6.31%. Treponema belonging to the phylum Spirochaetota can convert H₂ and CO₂ into acetic acid [38], corresponding to a lower pH value in S2, S4, and S5. This result agreed with the previous analysis. Both Caproiciproducens and norank_f__Ethanoligenenaceae belong to the phylum Firmicutes. Their relative abundances in S4 and S5 were higher than those of CK, and this situation persisted until day 30. Caproiciproducens can convert sugars into H₂ to provide an energy supply for hydrotrophic methanogens [39,40]. On day 50, the relative abundance of HN-HF0106 in CK was higher than that in S2, S4, and S5, indicating that SMX residue inhibited the activity of this bacterial genus. In contrast, Ruminiclostridium displayed a higher adaptability to SMX residue. Ruminiclostridium belonging to the phylum Bacteroidota can synthesize cellulose-degrading enzymes to decompose lignocellulose. On day 50, the easy-to-degrade organic matter had already been depleted, and the bacteria using difficult-to-degrade organic matter as the main nutrient supply prevailed.

Figure 8

Effects of nanoparticles on structural changes of the bacterial community involved in the anaerobic digestion of SMX-containing chicken manure on the genus level.