Identification and Classification of the Dissolved Substances from Sludge Biochar and Their Effects on the Activity of Acid Phosphomonoesterase

Abstract

Soil extra-cellular enzymes are the main driving force for microbial and biochemical processes, which makes them sensitive indicators for soil health and quality. Returning large amounts of sludge or its biochar to farmland may introduce exogenous substances into soil and have a significant impact on soil enzymatic activity. This study aimed to evaluate the effects of substances dissolved from sludge biomass and its biochar added at different amounts and produced at various temperatures (200 °C, 300 °C, and 450 °C) on the activity of acid phosphomonoesterase. Results showed that the activity of acid phosphomonoesterase was significantly inhibited by these dissolved substances from biochar pyrolyzed at different temperatures, especially at high concentrations of 50 mgC L⁻¹ and upon the exposure to DBC200. The conformation of acid phosphomonoesterase became loose and flexible after exposure to dissolved organic matter (DOM) extracted from biochar in terms of reduced α-Helix contents and increased β-Turn contents as deduced from circular dichroism spectra. According to the results of multiple linear regression, it can be concluded that the increased contents of arsenic as well as protein-like components within dissolved substances may be responsible for the inhibited enzymatic activities and the altered enzymatic conformation. Our findings provide evidence that the pyrolysis of sludge at a higher temperature would be helpful to reduce its negative impacts on the soil ecosystem.

1. Introduction

Sludge is an inevitable major byproduct of sewage treatment that is difficult to manage because it contains a lot of organic debris, bacteria, inorganic particles, and colloids [1]. With the accelerating process of human urbanization, the output of sludge is also increasing rapidly, the recycling of which creates a lot of problems. According to statistics, the annual output of sludge in the 35 major European countries was about 2,841,735 tons in 2018 [2]. If not properly disposed, such considerable amounts of waste will further threaten the environment and even human health. Pyrolysis has attracted more and more attention in recent years as an efficient waste treatment method. This is because pyrolysis can not only eliminate pathogens in sludge [3], reduce the production of dioxin [4], and fix most heavy metals in solid residues to inhibit the formation of secondary pollution [5], but also enables to produce bio-oil, non-condensable gas, and biochar with high utilization value [6]. This provides a new and effective way for sludge reduction which is harmless and allows for efficient resource utilization. Khadem et al. [7] found that soil biochar applications can greatly reduce the use of chemical fertilizers and promote the development of the sustainable agriculture owing to the stimulation on microbial activity, enzyme production, and activity. Some studies reported that sludge biochar can increase crop yields and reduce diseases and insect pests when applied for the purpose of soil improvement [1]. Other scientists showed more concerns regarding the risks associated with the application of sludge biochar [8]. Because limited studies with even contradictory results have been reported, and the biological effects of sludge biochar in these studies have been linked with its properties [9]. To properly process and manage sludge biochar, it is thus very necessary to assess its potential effects on soil properties before use in massive quantities.

Soil extra-cellular enzymes such as hydrolase hold a potential as sensitive indicators of soil health and quality owing to their direct or indirect participation in the material cycle and the functioning of soil ecosystems [10]. Among the soil extra-cellular enzymes, phosphomonoesterases have been studied most and their enzymatic reaction has been shown to proceed through hydrolysing monoester bonds mononucleotides and sugar phosphates to produce available phosphorus nutrients for plant utilization. Similar to most hydrolases, the activities of phosphomonoesterases depend on several factors such as soil properties, interactions with soil organisms, plant cover, leachate inputs, and the presence of inhibitors and activators [11]. The dissolved substances of sludge biochar such as (heavy) metals, polycyclic aromatic hydrocarbons (PAHs), phenols, and dissolved organic matter (DOM), which have a better mobility and a greater chance of contact with soil free hydrolases, can be a source of inhibitors for phosphomonoesterases that are readily dissolved in soil pore water as well. For instance, arsenic (As) may bind to cysteines in proteins and change the conformation and functions of those proteins [12], while purified Aldrich humic acid can bind with the protein lysozyme or even form encapsulated proteins and modify the protein structure [13]. Moreover, the pyrolysis temperature can strongly affect the composition and concentration of dissolved components of biochar. Normally, the total or available contents of N, P, and K are elevated as the pyrolysis temperature increases, while the amounts of DTPA-extractable Cu, Pb, Zn, Cd, and Cr are reduced due to the increased pH value or enhanced adsorption capacity of biochar [1,5]. Various components in biomass such as hemicellulose, cellulose, and lignin would decompose and produce different products over different temperature ranges [14]. It thus seems necessary to further understand how the dissolved substances released from sludge biochar produced at different temperatures interfere with the biological activity of phosphomonoesterases.

The present study therefore aims at investigating the biochar produced from sewage sludge, characterizing the dissolved substances (i.e., DOM, (heavy) metals) as a function of the pyrolysis temperature, and elucidating the mechanisms by which these dissolved substances affect the activity of purple acid phosphomonoesterase that was mostly secreted from bacteria and fungi [15,16] and generally prevails in acidic soil [17]. The first hypothesis of this study was that the activity of acid phosphomonoesterase would be inhibited by the dissolved substances released from sludge biomass or its biochar. In addition, He et al. [18] have reported that the DOM content released from dewatered sludge biochar decreased with increasing pyrolysis temperatures (200–600 °C). Due to the decreased contents of dissolved substances from sludge biochar, the degree of inhibition on the activity of acid phosphomonoesterase was thus hypothesized to decrease with the increase of pyrolysis temperature for sludge biomass and its biochar. This formed the second hypothesis of the present study.

2. Materials and Methods

2.1. Preparation and Characterization of Sludge Biomass and Biochar

The biochar used in the present study was produced from sewage sludge, which was obtained from a pharmaceutical company in Yunnan, China. It was previously dried at 50 °C after removing the large residues, ground in a laboratory mill, sieved to <0.150 mm particle size, and mixed evenly. The prepared sludge biomass (SB) was then pyrolyzed in a muffle furnace (Beijing Ever Bright Medical Treatment Instrument Company, Beijing, China) with N2. As reported by some researchers, sewage sludge biochar produced at 450 °C should be more suitable for soil improvement since it was less toxic to the growth of plants owing to the lower level of heavy metals, and the relatively moderate pH or ash levels within biochar [19,20]. Coupled with the consideration of reducing economic investment in recycling sludge, the pyrolysis temperatures for biochar production in the present study were therefore set at 200 °C, 300 °C, and 450 °C for 2 h. After cooling to room temperature, all biochar (BC200, BC300, BC450) was removed, ground, sieved to <0.074 mm, and stored in brown glass bottles at 4 °C for further use.

The pH values of SB, BC200, BC300, and BC450 were determined in deionized water at the ratio of 1:5 wt/v by a PXSJ-216F pH meter (Leici Instruments, Shanghai, China). The total mass percentages of C, H, O, N, and S were measured by a Vario MICRO cube elemental analyzer (Elementar, Hanau, Germany). The Brunauer–Emmett–Teller surface area was measured via the N2 adsorption multilayer theory using an ASAP2020M surface area analyzer (Micromerities, Norcross, GA, USA). The surface morphology was observed by a Quanta 200F scanning electron microscopy (FEI NanoPorts, Portland, OR, USA). All samples (50 mg) were digested by a mixture of HNO₃ (3 mL), HF (1 mL), and H₂O₂ (1 mL) in a sealed Teflon vessel. The concentrations of Mn, Cr, Fe, Cu, Zn, Ni, As, and Cd in all samples (i.e., SB, BC200, BC300, and BC450) were determined using a NexION 350x inductively coupled plasma mass spectroscopy (ICP-MS, Perkin Elmer, Waltham, MA, USA). The yield was determined by the ratio of the remaining mass of the sample after pyrolysis to the original mass of biomass. Each sample was analyzed in duplicate.

2.2. Extraction and Characterization of Dissolved Substances

To extract dissolved substances from biochar, 2.5 g of each biochar (i.e., SB, BC200, BC300, and BC450) was mixed with 50 mL of deionized water and then placed in a thermostatic oscillator (25 °C, 200 r·m⁻¹) for 24 h. After that, the samples were taken out and centrifuged at 1007× g for 30 min. Biochar particles were then removed by a 0.45 μm membrane filter (ANPEL Laboratory Technologies, Shanghai, China). The chemicals present in the supernatant were regarded as the dissolved substances (i.e., DSB, DBC200, DBC300, and DBC450) that were released from SB, BC200, BC300, and BC450. The supernatant was stored at 4 °C for further use. To determine the subsequent exposure concentration ranges, the total dissolved organic carbon (DOC) content of each sample was measured using the Vario MICRO cube elemental analyzer, and the samples were used to prepare different concentrations of DSB, DBC200, DBC300, and DBC450. The metal concentrations of Mn, Cr, Fe, Cu, Zn, Ni, As, and Cd in each sample of dissolved substances were determined by ICP-MS (Perkin Elmer, Waltham, MA, USA). The concentrations of PAHs and phenolic compounds were determined by Yunnan Province Geology and Mineral Environmental Monitoring Center, China. Sixteen PAHs [21] were analyzed using a LC 2030 high performance liquid chromatography (SHIMADZU, Beijing, China), and thirteen phenolic compounds [22] were analyzed using 8890 gas chromatography-mass spectrometry (Agilent, Palo Alto, CA, USA).

Fluorescence excitation-emission matrix (EEM) spectroscopy coupled with parallel factor analysis (PARAFAC) was used to better understand the compositional features of DSB, DBC200, DBC300, and DBC450 including the dominant fluorescent components based on their excitation and emission (Ex/Em) maxima. The EEM of each sample with DSB, DBC200, DBC300, and DBC 450 was generated from 250 nm to 600 nm at 1 nm intervals, with 5 nm increments of the excitation wavelength from 220 nm to 400 nm using a F-7000 fluorescence spectrophotometer (HITACHI, Tokyo, Japan). For the EEM analysis, the concentration of C in each sample was diluted to 1 mgC L⁻¹ to eliminate the inner-filter effect of fluorescence. The detailed method of using PARAFAC (EFC v1.2, He wei, Beijing, China) to model the dataset of fluorescent EEMs was described in the studies of Rajapaksha et al. [23] and Li et al. [24].

2.3. Exposure Testing Design

Because the hydrolysis of p-nitrophenyl phosphate (pNPP) is much more rapid and accurate than that of natural substrates such as nucleic acids [11]. The pNPP assay was selected to show the activity of purple acid phosphomonoesterases with or without the dissolved substances released from biochar. The purple acid phosphomonoesterase (from potato) was used in this study as its molecular structure has been well investigated [25]. Both purple acid phosphomonoesterase at a nominal concentration of ≥0.5 unit/mg solid and pNPP (10 mM) were purchased from the Sigma-Aldrich Company, St. Louis, MO, USA. The optimum pH value for acid phosphomonoesterase activity was determined using the dynamic light scattering method by a Zeta PALS instrument (Brook Haven, Attleboro, MA, USA) when the surface charges of the acid phosphomonoesterase and pNPP were opposite. This is because the combination of substrate and enzyme by electrostatic attraction is the primary condition for enzymatic reactions [26]. Upon the pre-experiment basis, the solution pH value was thus kept around 3 to avoid the influence of pH alterations and to ensure the combination of substrate (i.e., pNPP) and enzyme (i.e., acid phosphomonoesterases). The pNPP is hydrolysed to p-nitrophenol (pNP), the content of which can be determined spectrophotometrically at 405 nm of a LD-SY96S automatic microplate spectrophotometer (Laiende, Kunshan, China). The pNP standard solution purchased from the Sigma-Aldrich Company was used to establish the standard curve, i.e., the linear correlation between the pNP concentration and the absorbance, so as to facilitate the later confirmation of the product concentration.

Acid phosphomonoesterase was dissolved in ultra pure water to prepare a stock solution of 0.05 unit mL⁻¹. The original solutions of DSB, DBC200, DBC300, and DBC450 were diluted several times using ultra pure water, 4 mL of which were added into a 10 mL brown vial and then mixed with 4 mL acid phosphomonoesterase to obtain the dissolved substances concentrations of 0, 5, 12.5, 25, and 50 mgC L⁻¹. The pH value of the spiked samples was adjusted to around 3 using either 0.1 M HCl or NaOH. Samples were then sealed and placed on a shaker at 80 rpm and 25 °C for 2 h. The activity of acid phosphomonoesterase was then detected using Acid Phosphatase Assay Kits (Beyotime bio-technology company, Shanghai, China) according to the protocol of the manufacturer. All treatments were repeated three times. The Chirascan V100 circular dichroism (Applied Photophysics Limited, Surrey, UK) and the Beta Structure Selection method (BeStSel) were used to explain the conformation changes [27] of acid phosphomonoesterase before and after its exposure to DSB, DBC200, DBC300, and DBC450.

2.4. Statistical Analysis

The activity of acid phosphomonoesterase was determined by the generated pNP content which was derived from the standard curve (see Figure S2) and was expressed as nmol of pNP generated per minute. The degree of inhibition of the enzymatic reaction was calculated by means of Equation (1):

$$\mathit{Inhibition}\mathit{rate}=(1-\frac{\mathit{generated}\mathit{pNP}\mathit{of}\mathit{treatment}\mathit{groups}}{\mathit{generated}\mathit{pNP}\mathit{of}\mathit{control}\mathit{groups}})\times 100\%$$

(1)

The normal distribution of data was verified by the Kolmogorov–Smirnov (K-S) test. One-way analysis of variance (ANOVA) was carried out to detect the significant level of differences between percentages of C1, C2, and C3 within DSB and those percentages within DBC200, DBC300, or DBC400 treatments using the Least Significant Difference method (LSD). The significant level of differences in inhibition rates of phosphatase activity was also analyzed using the one-way ANOVA method. The multiple linear regression method was used to determine the main contributing factors towards the inhibition on enzyme activity. The characterization data of dissolved substances were all fitted to the multiple linear regression model, and then the parameters with significant correlations (p < 0.05) were left in the final formula. The data included the measured metal concentrations of Mn, Cr, Fe, Cu, Zn, Ni, As, and Cd; the maximum fluorescence intensities (Fmax) of the three main components, namely C1, C2, and C3; the concentrations of 16 PAHs or ΣPAH; the concentrations of 13 phenolic compounds; and the DOC concentrations. The statistical analysis was conducted using the software package SPSS 21 (IBM Corporation, Amonk, NY, USA) and the plotting was conducted using the OriginPro 8 software (Origin Lab, Northampton, NY, USA).

3. Results and Discussion

3.1. Biochar Yield and Characterization for Particles and Dissolved Substances

As summarized in

Table 1. Properties of sewage sludge biomass (SB) and biochars pyrolyzed at 200 °C (BC 200), 300 °C (BC 300), and 450 °C (BC 450).

Properties	SB	BC 200	BC 300	BC 450
Biochar yield (%)	-	88.7 ± 3.8	78.9 ± 2.1	70.5 ± 2.4
pH	8.5 ± 0.4	8.0 ± 0.3	8.3 ± 0.2	9.2 ± 0.3
Specific surface area (m2 g−1)	8.7	6.0	15.6	35.0
C (%)	-	26.4	23.1	22.0
H (%)	-	3.4	2.7	1.7
O (%)	-	26.0	24.4	23.2
N (%)	-	2.0	1.8	1.2
S (%)	-	0.47	0.51	0.62
H/C	-	1.6	1.4	0.8
O/C	-	0.80	0.79	0.80
(O+N)/C	-	0.87	0.86	0.85
Mn (mg kg−1)	-	697.4 ± 32.8	752.1 ± 36.6	925.7 ± 44.2
Cr (mg kg−1)	-	341.3 ± 16.1	391.0 ± 18.3	416.7 ± 20.1
Fe (mg kg−1)	-	6515.3 ± 225.7	16,241.4 ± 349.3	23,123.1 ± 756.1
Cu (mg kg−1)	-	269.4 ± 12.4	206.8 ± 9.34	295.5 ± 12.7
Zn (mg kg−1)	-	6402.9 ± 220.1	15,884.5 ± 594.2	13,267.0 ± 463.3
Ni (mg kg−1)	-	21.9 ± 0.9	89.9 ± 3.49	36.6 ± 1.2
As (mg kg−1)	-	11.5 ± 0.3	25.3 ± 0.8	41.4 ± 1.3
Cd (mg kg−1)	-	2.0 ± 0.1	3.7 ± 0.1	4.2 ± 0.1

- denotes not determined.

, the pH values of SB, BC200, BC300, and BC450 were all in the alkaline range. As the pyrolysis temperature increased, the total concentrations of Mn, Cr, Fe, Cu, Zn, Ni, As, and Cd continually increased (Table 1), whereas the dissolved fractions of those metals decreased (Table S2). This result was consistent with the previous studies by Bai et al. [28] and Li et al. [29], indicating a further decrease in bioavailability, leachability, or environmental risks of metals in biochar. Due to the removal of volatile materials [23] as well as the appearance of loose structure and fine pores (see Figure 1), the specific surface area of biochars was increased by increasing pyrolysis temperatures. A lower molar ratio of H/C at relatively higher temperatures (i.e., 300 °C and 450 °C) indicated a more carbonized biochar as compared with the biochar produced at 200 °C, while the similar O/C ratios indicated that the hydrophilicity of biochar changed insignificantly in the present range of pyrolysis. In addition, the amount of DOC produced by SB and BC at similar mass contents increased first with increasing temperatures, and then decreased sharply by more than 50% at 450 °C (see Table S2). This may be because the decomposition temperature varied for different organic matters in sludge biomass. For example, hemicellulose began to decompose at the range of 220–315 °C, cellulose began to decompose from 300 to 400 °C, and lignin can decompose from 150 to 900 °C [14]. In addition, some volatiles in the biochar may be secondary cracked and escaped when the temperature was raised [23,30], leading to the reduced amount of DOC at a higher pyrolysis temperature.

Using the EEM-PARAFAC method (see Figure 2 and Figure S1), three similar fluorescent components including two humic-like substances (C1 and C2) and one protein-like substance (C3) were identified from the dissolved substances of sludge biomass and its biochar (see

Table 2. Characteristics and sources of the major components of dissolved substances (DSB, DBC200, DBC300, and DBC450) identified by the EEM-PARAFAC analysis.

Component	Ex (nm)	Em (nm)	Fluorescent Compound	References
DSB
C1	250/335	410	Humic-like	[34]
C2	240/405	470	Humic-like	[31]
C3	225/280	336	Protein-like (tyrosine)	[35]
DBC200
C1	220/335	410	Humic-like	[36]
C2	250/370	460	Humic-like	[34]
C3	225/285	350	Protein-like (tryptophan)	[37]
DBC300
C1	225/320	390	Humic-like (aliphatic carbon)	[36]
C2	225/365	438	Humic-like (fulvic acid)	[38]
C3	225/280	304	Protein-like (tyrosine)	[23]
DBC450
C1	225/320	390	Humic-like	[36]
C2	220/365	402	Humic-like (fulvic acid)	[38]
C3	225/280	310	Protein-like (tryptophan)	[39]

). Kothawala et al. reported that C1 was described as a high molecular weight aromatic and C2 was described as a low molecular weight substance [31]. As shown in Figure 3, more than 50% of the dissolved substances of the sludge biomass and its biochar were dominated by humic-like substances (i.e., C1 and C2). With the increase of pyrolysis temperature, the relative abundance of C1 and C2 increased significantly, while the relative abundance of C3 decreased significantly. This was deduced through comparison between the sum of C1 with C2 of DSB (63%) and the values of DBC200 (79%), DBC300 (87%) or DBC450 (90%) using the one-way ANOVA analysis. This demonstrated an essential role of pyrolysis temperature in affecting the properties of dissolved substances from sludge biomass and its biochar. The increased pyrolysis temperature may promote the decomposition of (poly)phenolics and other aromatic structures in biochar, leading to the increased relative abundances of C1 and C2. This was consistent with the results found by Rajapaksha et al. [23] and Lin et al. [32]. In contrast, the reduced relative abundance of C3 may indicate a poor heating stability of protein-like substances (e.g., amino acids and peptides [33]) in the dissolved substances of sludge biomass and its biochar.

3.2. Activity of Acid Phosphomonoesterase as Affected by Dissolved Substances

As shown in Figure 4, the presence of dissolved substances from sludge biomass and its biochar did inhibit the activity of acid phosphomonoesterase, i.e., the calculated inhibition rates ranged from 11.9% to 91.5%. This finding confirmed our first hypothesis. At higher concentrations of DOC (i.e., 25 and 50 mgC L⁻¹), the highest inhibition rate on activity of acid phosphomonoesterase was observed in DBC200 which was significantly higher than the inhibition rate in DSB. The lowest inhibition rate was detected in either DBC300 or DBC450 (see Figure 4B). At lower concentrations of DOC (i.e., 5 and 12.5 mgC L⁻¹), the maximum inhibition rate occurred in DSB and DBC200. This rate was significantly higher than in the treatments of DBC300 and DBC450. This finding partially allowed us to accept our second hypothesis of the degree of inhibition of dissolved substances from sludge biomass and its biochar on the activity of acid phosphomonoesterase decreasing with increasing pyrolysis temperature. In addition, a dose-dependent trend was observed for DBC200 inhibition on the activity of acid phosphomonoesterase (see Figure 4A), while application of BC300 at a dose of more than 25 mgC L⁻¹ significantly increased the inhibition on the activity of acid phosphomonoesterase as compared with the treatments at a dose of 5 mgC L⁻¹. This showed that the contents of inhibitors for enzyme activity were enhanced with the increasing concentrations of dissolved substances from sludge biomass or its biochar.

Data shown in Table 1 and Table S1 were all fitted to the multiple linear model (see Section 2.4). Factors left in Equation 2 indicated a statistically significant contribution in influencing the inhibition rate on the activity of acid phosphomonoesterase. According to previously reported studies, pH, (heavy) metals, DOM, phenolic compounds, and PAHs can alter the enzyme activities in the presence of biochar [9,40]. However, in the present study, the pH value of the system was fixed around 3 during the enzyme activity testing. The shift of pH values induced by the addition of dissolved substances from biomass or biochar can be excluded as a reason for the alterations in acid phosphomonoesterase activities.

$$\begin{array}{c}\mathit{Inhibition}\mathit{rate}=15.331\times C_{\mathrm{As}}+0.034\times F_{\mathrm{maxC3}}-0.003\times F_{\mathrm{maxC1}}-5.581\\ R^2=0.95,p<0.05\end{array}$$

(2)

where C indicates the concentrations of metals in the dissolved substances of sludge biomass or its biochar, μg L⁻¹; F_max indicates the measured peak value of the corresponding component in the dissolved substances of sludge biomass or its biochar using the EEM spectroscopy, a.u.

The above results of multiple linear regression (R² = 0.95, p < 0.05) showed that there were statistically significant positive relationships between inhibition rates of enzymatic activity and the concentration of As or the measured peak value of C3 within the dissolved substances of sludge biomass and its biochar. This indicated that only soluble As and protein-like components within the dissolved substances were contributing drivers for the reduced enzyme activities in the present study. The significantly negative relationship that occurred between the inhibition rate and the measured peak value of C1 indicated that the humic-like component had a protective effect on acid phosphomonoesterase. From this aspect, the second hypothesis can be only partially accepted because the induced negative effects on enzyme activity were dependent on the component types within the dissolved substances from biomass and biochar. The previous study reported that HAsO₄²⁻ may compete with PO₄³⁻ for binding at the active sites of acid phosphomonoesterase and thus inhibit the activity of potato acid phosphatase [41,42]. The protective effects of humic-like substances (C1) within the dissolved substances of sludge biomass and its biochar were postulated to originate from their strong complexation with metal ions [43] or oxidizing As (III) to As (V) to reduce its negative effects [42]. As depicted in Figure 3, although C1 accounted for 49% of the PARAFAC components in DBC200 which was similar to the C1 contents in either DBC300 or DBC450, the inhibition rate was still significantly higher than the rate observed for DBC300 and DBC450. This indicated a weak contribution to inhibition rate from C1. As a comparison, DBC300 and DBC450 with much lower C3 proportions did produce a much lower degree of inhibition rate of acid phosphomonoesterase. This may be attributed to conjugate interactions between biomolecules such as tyrosine or tryptophan [44,45] in solution, leading to structural abnormalities in the enzyme, as further discussed in Section 3.3.

3.3. Conformation of Acid Phosphomonoesterase as Affected by Dissolved Substances

According to BeStSel, the circular dichroism spectra showed a significant decrease of the α-Helix structures accompanied by a significant increase of β-Turn structures in acid phosphomonoesterase exposed to DSB, DBC200, DBC300, and DBC450 (see

Table 3. The influence of dissolved substances (DSB, DBC200, DBC300, and DBC450, 12.5 mgC L⁻¹) released from sludge biomass and its biochar on the secondary structure of acid phosphomonoesterase.

Estimated Secondary Structure Content (%)	Enzyme	DSB	DBC200	DBC300	DBC450
$\mathsf{\alpha }$-Helix	13.9	0.0	0.0	0.0	0.6
$\mathsf{\beta }$-Sheet	28.1	30.9	31.7	32.1	33.0
$\mathsf{\beta }$-Turn	5.2	13.1	13.7	13.8	11.6
$\mathsf{\alpha }+\mathsf{\beta }$	52.8	56.0	54.5	54.1	54.8

). A pyrolysis temperature-dependent trend was unfortunately not observed in the conformation changes of acid phosphomonoesterase. As shown in Table 3, the missing α-Helix contents indicated that the acid phosphomonoesterase became loose or even unfolded after the exposure to DSB, DBC200, DBC300, and DBC450. This was likely caused by the dissociation of α-Helix hydrogen bonds other than complexation between As (III) and sulfhydryls [12] because there were no cysteine residues, acetylcysteine residues, or homocysteine residues which may consist of sulfhydryls in the α-Helix structure of acid phosphomonoesterase [25]. The increased β-Turn contents revealed an extremely flexible structure of acid phosphomonoesterase after exposure, which makes the enzyme molecule move freely and allows it to cover its active sites for substrate binding [46]. Due to the complexity of DSB, DBC200, DBC300, and DBC450, it was still difficult to accurately quantify the contributions of As, C1, or C3 on enzymatic inhibition or conformation change in the present study. Our findings did indicate that the secondary structures of acid phosphomonoesterase was very sensitive to dissolved substances of sludge biomass and its biochar.

4. Conclusions

In summary, our findings provided evidence that the dissolved substances released from sludge biomass and its biochar significantly reduced the activities of acid phosphomonoesterase, while the inhibition degree declined with increasing pyrolysis temperatures. The reduced enzyme activities were accompanied by structural changes in the α-Helix and β-Turn contents of acid phosphomonoesterase and were likely explained by the increased contents of arsenic as well as the protein-like components within the dissolved substances from the sludge biomass and its biochar. The present study reflected an in-depth analysis in the soil enzymatic burden of dissolved substances from sludge biomass and its biochar, also suggesting that increasing pyrolysis temperature was very critical for obtaining a sludge biochar with less impact on soil functions. Therefore, we suggest using the sludge biochar produced at a higher temperature (>450 °C) and focusing on getting a clearer map of sludge biochar–enzyme interactions across different scenarios in future investigations.