Dephenolization pyrolysis fluid improved physicochemical properties and microbial community structure of saline-alkali soils

Saline-sodic soil is widely distributed around the world and has induced severe impacts on ecosystems and agriculture. Biomass pyrolysis fluid (BPF), as a substance rich in organic acids, has been proposed as a saline-alkali soil conditioner. One of the main problems with BPF applications is the potential contamination of the phenolic substances it contains. Therefore, the purpose of this study is to reduce the phenolic substances in BFP and study the improvement effect of BFP on saline-alkali soil. Firstly, we explored the physicochemical properties of BPF prepared at different temperatures (300 °C, 400 °C, 500 °C, 600 °C, and 700 °C). Then BPF was separated into upper phases (UP) and lower phases (LP) by a simple one-step salting-out extraction method. We found that phenolic substances were mainly concentrated in the UP (average content was 193.27 mg/g), and the content of phenolic substances in the LP was effectively reduced (average content was 64.52 mg/g). Next, we added the LP diluted at different times (0, 50, 100, 200, 400) into saline-alkali soil for improvement experiments. The experimental results show that the lower phase diluted 400 times at the pyrolysis temperature of 500℃ was added into saline-alkali soil, which greatly increased the content of soil available nutrients. Under the action of organic acids, soil pH (the average was 7.43) and total salt content could be reduced effectively, and soil enzyme activities can be increased. Microbial community analysis showed that the addition of LP could increase the proportion of Actinomycetes, which played a beneficial role in improving soil fertility and then improved the growth of Chinese cabbage.


Introduction
Soil salinization and alkalization were global problems restraining the improvement of land-use capability and the development of agricultural production (Na et al. 2015). It has been reported that over 1.1 billion hectares of land, involving more than 100 countries and regions, are affected by soil salinity ). On the one hand, soil salinity affected the growth of crops and exacerbates the crisis of food shortages; on the other hand, it caused an annual economic loss of at least 27.2 billion dollars in agriculture production all over the world (Kou et al. 2019). Thus, the improvement and utilization of salinized land have gradually attracted people's extensive attention. The restoration of saline soils has been extensively studied by many researchers in terms of theory, measures, and evaluation methods (Soldo et al. 2020). Physical, chemical, and biological methods have also been extensively studied, for example, artificial irrigation (Lei et al. 2019), salt-tolerant plants (Jin et al. 2017), compost, and manure. However, these measures were costly and difficult to apply in practice on a large scale.
It was well known that biomass (i.e., wood and straw) was a natural widespread renewable resource (Laëtitia et al. 2019). Biomass, under conditions of oxygen isolation, was pyrolyzed at high temperatures to produce a liquid called pyrolysis fluid (BPF) (Ys et al. 2020). Due to its high content  (Mamaeva et al. 2016), it has been used by some researchers to study saline soil improvement. It was reported that the diluted BPF could reduce the pH and soil salt in salinized land, improve soil organic matter mass fraction, significantly increase the number of soil microorganisms and promote plant growth (Xinyan et al. 2018). Despite the positive effect of BPF on saline land improvement, a large number of phenolic substances exist in BPF. Phenol and its derivatives are aromatic compounds and a kind of protoplasmic poison, which can produce toxicity to all biologically active bodies (Avnish et al. 2021). Therefore, the refinement of BPF becomes an important condition for its better utilization. Currently, conventional distillation ), molecular distillation (Liu et al. 2020), and graded condensation (Moutsoglou et al. 2018) have been investigated as the main BPF refining methods. However, these methods were expensive and required additional energy consumption. Salting-out extraction (SOE) was a separation method used to extract hydrophilic product from its aqueous solution with the aid of inorganic salt as the salting-out reagent and organic solvent as the extractant (Wang et al. 2014). As a novel, easy-to-use, low-cost and low-energy method, it has been used by several researchers in the field of extraction and purification of organic substances. Fu et al. demonstrated that a salting-out extraction system composed of monosodium phosphate and ethanol exhibited excellent extraction efficiency for both butyric acid (∼99%) and acetic acid (∼90%) (Fu et al. 2017). However, SOE has not been used for the refinement of BPF. Therefore, the purpose of this study was to investigate the refining effect of SOE on BPF. Furthermore, the effect of refined BPF on saline land improvement was evaluated by changes in soil physicochemical properties, enzyme activity, available nutrient content, and microbial community. Besides, we further verified the role of BPF in the restoration of saline land by growing various biological indicators of cabbage.

Material preparation
The cotton stalk (CS) was picked from the experimental agricultural farm of Shihezi University and air-dried in the ventilation area. The CS was cut into small pieces with a particle size of 2 cm, dried in an oven at 105 °C for 4 h, and then placed in a desiccator for standby use.
In this experiment, the saline-alkali soil (white alkali saline-alkaline soil) was taken from the vicinity of Mushroom Lake Reservoir in Shihezi City (85°9′39″E, 44°4′75″N), and sampling points were set in the sampling area in the S-shaped. The soils were collected from the surface layer (0-20 cm). Before the start of the test, the soil was air-dried and passed through a 3-mm sieve. The basic physicochemical properties of soils were shown in Table 1.

Preparation of biomass pyrolysis fluid
In this study, the biomass pyrolysis fluid (BPF) was obtained in the process of making biochar from the CS, 100 ± 0.5 g CS was loaded into the tube, and N 2 with a flow rate of 300 mL/min was purged into the furnace. Then the furnace was heated from ambient temperature to different temperatures (300, 400, 500, 600, and 700 ℃) with a heating rate of 10 °C/min and held for 60 min to ensure the complete pyrolysis. BPF was collected during the pyrolysis using a circulating water condensation system. After precipitation and filtration of the collected condensate, the brown and clear liquid BPF was obtained. It should be noted that the BPF was loaded into a brown glass bottle and stored in a refrigerator at 4 °C. Each trial was performed 3 times and all BPF was subsequently analyzed.

Salting-out extraction
The saturated ammonium sulfate solution was allocated and added to the centrifuge tube, and ethanol was added to mix with BPF (The mass ratio of ammonium sulfate solution, BPF, and alcohol was 6:6:1). After shaking, ultrasonic processing was carried out for 15 min, followed by centrifugation in a frozen centrifuge at 8000 rpm/min at room temperature for 15 min. After standing for some time, the upper phases (UP) and lower phases (LP) were separated. The above experiment was repeated three times.

Pot experiment
In this pot experiment, the samples used for irrigation were ultrapure water (CK) and LP with different pyrolysis temperatures and different dilution multiples (50, 100, 200, and 400). Before starting the experiment, 250.0 g of saline soil was placed in each pot. The soil surface was loosened before sowing and the seeds were spread evenly in the pots, then about 3 mm of soil was spread on the surface of the rapeseed, the pots were covered with plastic wrap and all pots were placed in the greenhouse. The pots were irrigated separately with different samples every 3 days, with 30 mL of irrigated samples per day for the first 10 days and 50 mL of irrigated samples per day for the last 25 days. The germination of each pot of cabbage was recorded 3 days after sowing, and the two seedlings with the worst growth rate were pulled out every 7 days to measure plant height for a 35-day trial period. pH and total soil salinity, as well as enzyme activities such as urease and sucrase, and soil organic matter and available nitrogen, phosphorus, and potassium were measured at the end of the potting cycle.

Analysis and characterization of BPF
The determination of BPF density is determined by the pycnogenol method. Accurately measure 10 mL of the solution to be measured and put it into a density bottle and store it at a constant temperature (20 °C). Weigh precisely 10 mL of the mass of the liquid to be measured on a balance, the density of which can be obtained by dividing the mass by the mass. The water content was determined using a Karl Fischer moisture meter (787KF, Metrohm, Swiss). The pH value of BPF was determined using a pH meter (MP551, Shanghai San-Xin Instrumentation, Shanghai, China). The determination of total polyphenols (TP) content of the analyzed samples was assessed using the Folin Ciocalteu reagent (Luque-Rodríguez et al. 2007). The absorbance was measured at 765 nm (8453UV, Agilent Technologies, Santa Clara, CA, USA). Polyphenol content was calculated from the calibration curve plotted using gallic acid as the reference standard. The results are shown as mg of gallic acid in 1 L of liquid (mg GAE/1 L). All assays were performed in triplicate. The determination of total acid (TA) of the BPFrefers to the national standard GB/T 12,456-90, which is determined by the titration method, and the total acid is calculated by acetic acid. Using 0.1 mol/L of NaOH solution titration, 3-4 drops of 10 g/L of phenolphthalein solution are added to the solution to be measured, and the color of the solution to be measured changes from orange-yellow to orange-red, which can be regarded as reaching the titration endpoint.
The organic composition of BPF was analyzed by GC-MS (GCMS-QP2020, Shimadzu, Japan). The GC-MS condition was: the column was "HP-INNOWAX (30 m × 0.25 mm × 0.25 μm)." Helium gas as a carrier gas, carrier air velocity of 1 mL/min. The injection temperature was 260 °C. The shunt ratio was 20:1. The column temperature was maintained at 50 °C for 3 min, heated to 250 °C at 4 °C/min, and maintained for 15 min. The NIST2011 mass spectrometry library was used for qualitative analysis.

Soil nutrient and enzyme activity analysis
Urease, Sucrase, catalase, and alkaline phosphatase activity were measured by sodium phenol-sodium hypochlorite colorimetric method, 3,5-dinitrosalicylic acid colorimetric method, potassium titanate titration method (Tapia et al. 2016) and colorimetric method of benzyl phosphate (Mishra et al. 2014), respectively. The alkaline diffusion method (Leng et al. 2020), 0.5 mol/L NaHCO 3 methods, NH 4 OAc, flame photometry, and volumetric method of potassium dichromate-dilution calorimetry (Musadji et al. 2020) were used to measure alkaline nitrogen, fast-acting phosphorus, fast-acting potassium and organic matter content in soil separately.

High-throughput sequencing
The diversity of microbial communities in soil was analyzed in the Illumina Misep sequencing platform (Illumina, USA) using the full-length 16S rRNA sequence . For microbiome samples from a variety of different sources, the most appropriate total microbiome DNA extraction method was selected according to the experience of previous projects, and the DNA extraction quality was detected by 0.8% agarose gel electrophoresis, and the DNA was quantified by ultraviolet spectrophotometer. Usually targeting target sequences such as microbial ribosome RNA that can reflect the composition and diversity of the microbial flora, corresponding primers are designed according to the conserved regions in the sequence, and sample-specific Barcode sequences are added, and then PCR amplification of variable regions of rRNA genes (single or consecutive multiple) or specific gene fragments. PCR amplification uses NEB's Q5 high-fidelity DNA polymerase and strictly controls the number of amplification cycles to keep the number of cycles as low as possible while ensuring consistent amplification conditions for the same sample. The PCR amplification product was detected by 2% agarose gel electrophoresis and the target fragment was recovered by cutting and recovering using AXYGEN's gel recovery kit. Referring to the preliminary quantitative results of electrophoresis, the PCR amplification recovery product was quant-iT PicoGreen dsDNA Assay Kit, and the quantitative instrument was a Microplate reader (BioTek, FLx800). Based on the fluorescence quantification results, each sample is mixed in proportion to its sequencing requirements. Taking Illumina MiSeq sequencing as an example, a sequencing library was prepared using Illumina's TruSeq Nano DNA LT Library Prep Kit (Bates et al. 2011).
The sequences were initially screened according to the following criteria and eliminated from further analysis: requiring sequence length ≥ 160 bp and not allowing ambiguous base N. We will exclude (1) sequences with primer mismatch base number > 1 at the 5′ end and (2) sequences containing consecutive identical base numbers > 8 (Tanja and Steven 2011). After chimera checking using USEARCH, UCLUST was used to cluster 97% sequence identity into an operable taxonomic unit (OUT) from which the alpha diversity indices (Simpson index) were determined. Principal component analysis (PCA) was used to assess the inter-relationships between different acclimatized microbial communities (Ramette et al. 2007).

Total phenol and total acid content in BPF, UP, and LP
The pH, density, yield, moisture content, and heating value of BPF were measured during the study period (see Supplementary Materials). When the pyrolysis temperature was low (300-400℃), the aliphatic hydroxyl groups on the branched chain of the lignin structure were broken, and the cellulose would depolymerize and dehydrate (Mi et al. 2016). The main products of their reactions were acetic acid, formic acid, other acid compounds, a small number of tar substances, and phenolic compounds (Ren et al. 2017). Therefore, as shown in Fig. 1a, the acid content was higher at a lower temperature, up to 139.23 mg/g. With the increase in pyrolysis temperature, the secondary pyrolysis of BPF was strengthened and the organic acids decomposed. For Fig. 1 a Content of TP and TA in BPF. b UP and LP percentage after salting-out extraction. c Content of TP in UP and LP. d Content of TA in UP and LP example, levoglucosan was decomposed into small molecular gas products through molecular reforming, decarboxylation, and other reactions, thus reducing the acid content (Ben et al. 2019). On the other hand, the increase in the total amount of BPF also led to a decrease in the proportion of acid compounds in the total organic matter. The higher the pyrolysis temperature was, the more intense the pyrolysis reaction of lignin was. Guaiacol (also known as o-methoxy-phenol), one of the main products of pyrolysis, and tar-like substances produced at low temperatures had secondary cracking. The GC-MS analysis of BPF showed that the content of guaiacol did decrease with the increase in pyrolysis temperature (see Supplementary Materials) . This led to an increase in the number of phenols and a rapid increase in TP content. In addition, the chemical bonds of oxygen-containing compounds in BPF broke in the order of C-O, C-H, and C-C bonds from weak to strong, and decarboxylation, decarbonylation, and dehydrogenation occurred respectively (Hoyong et al. 2018), so oxygen-containing compounds converted into more stable phenolic compounds. Therefore, as shown in Fig. 1a, TP reached a minimum of 67.27 mg/g at 300℃ and a maximum of 76.79 mg/g at 700℃.
From Fig. 1b and supplementary materials, it can be seen that the two-phase product obtained by salting-out extraction, UP was much smaller than the LP product, and the yield of LP product was first increased and then decreased, the highest yield was 96.29% at 500 ℃. The TP was mainly enriched in UP. The average TP content in UP was 193.27 mg/g, while that in LP was 64.52 mg/g (Fig. 1c). Because when inorganic salt solutions were added, the cations produced by ionization attract more water molecules to form a hydration layer, causing more organic molecules to be repelled into the organic phase (Xiao et al. 2009) (See supplementary materials). As shown in Fig. 1d, the effect of salting-out extraction on the TA of UP and LP in each temperature segment was the same as that of BPF, and the TA content in UP was slightly higher than LP after the saltingout extraction of BPF at each temperature. Therefore, in the subsequent pot experiments, we only studied the effect of LP on plant growth as and soil.

Effects of LP on plant growth
Compared with CK, LP had a significant effect on the germination rate (3 days), plant height (35 days), and plant weight (35 days) of Chinese cabbage (Fig. 2a-c). In particular, a 400-fold dilution of LP increased the germination rate by 25.76% on average, while a 50-fold dilution of LP only increased the germination rate by 2.4%. The plant average height and plant weight of Chinese cabbage, reached a maximum height of 6.7 cm and a maximum weight of 0.9661 g, both of which were higher than CK, by 67.51% and 93.22%, respectively. As the dilution increases the plants grew better (see Supplementary Material). When the dilution was low, the phenolic concentration in LP was higher, and the excessive phenolic compounds have an inhibitory effect on seed germination and plant growth (Williams and Hoagland 1982). On the other hand, the addition of sulfate, which increases the soluble salt content in the soil, will change the osmotic pressure in the plant, thus causing some hindrance to the plant root system for nutrient and water uptake. It even inhibits plant growth, while salt stress can cause ion toxicity in the plant body (Shahzad et al. 2021). This also results in severe plant senescence and high MDA content (Fig. 2d). Four 100-fold LP diluted in each temperature segment had a good effect on the plant's MDA, with an average decrease of 42.35% compared with the blank control group. Through pot experiments, we found that LP promoted plant growth to varying degrees. To further investigate the specific mechanism of LP, soil nutrients, enzyme activities, and microbial community structure was analyzed.

Effects of LP on soil nutrients and enzyme activities
The change in soil pH was inversely proportional to the TA content in LP (Fig. 3a). The salt used in the test was (NH 4 ) 2 SO 4 , which was a strong acid and weak base salt. Its solution was acidic, so it made a more significant impact on soil pH value. In addition, the addition of (NH 4 ) 2 SO 4 had a certain influence on soil total salt content (Fig. 3b) and alkali-hydrolyzable nitrogen content (Fig. 3d). Also, the addition of a high concentration of LP promoted the formation of macromolecule complexation in the soil, but it was not discharged in a short time (Shang et al. 2021). With the increase in the test period, small molecule salt was regenerated, leading to an increase in salt content. When using the appropriate dilution ratio (200 and 400 times), it could effectively complex salt ions and reduce the total salt content in the soil (Chen et al. 2019). LP was rich in organic materials, so when it was added to the soil, the larger the dilution ratio of LP was, the less the content of organic matter was in the soil. When LP was diluted by 50, 100, 200, and 400 times, the average organic matter content of soil increased by 66.75%, 47.16%, 29.39%, and 24.76%, respectively (Fig. 3c).
As shown in Fig. 3d-f, LP can significantly increase the effective soil nutrient content. LP contained a certain amount of acidic organic matter. On the one hand, the acid dissolution, chelation dissolution, and desorption of these acidic substances had an important and direct effect on the increase of soil nitrogen, phosphorus and potassium elements (Gautier et al. 2021). And small molecular acids could inhibit the mineralization of these elements and promote the release of available elements in soil . The migration of colloidal ions in soil was promoted by acidic ions and organic active molecules in LP. This resulted in the release of the original fixed nutrients in the soil and at the same time increased the activity of the original nutrients ). On the other hand, the acidic components of LP could interact with materials in the soil at the right concentrations. This could promote enzyme and microbial activity, thus further improving soil activity . Therefore, with the addition of LP, the contents of basic nitrogen, available phosphorus, and available potassium significantly increased, up to 139.56 mg/kg, 14.82 mg/kg, and 71.53 mg/g, respectively.
Soil colloids and minerals had adsorption effects on enzymes, this can reduce soil enzyme activity . LP contained a large number of acidic substances such as acetic acid, which can compete with soil colloids and minerals for adsorption sites on soil enzymes, thus reducing adsorption (Kabiri et al. 2016). From Fig. 4, it can be seen that LP had a significant promotion effect on soil enzyme activity. The urease and alkaline phosphatase activity increased with the increase of the dilution ratio of LP. Especially when the dilution ratio of LP was 200 and 400 times, the promotion of both enzymes was greater. Compared with CK, urease activity increased on average 3.13-fold (Fig. 4a). The trend graph of alkaline phosphatase activity (Fig. 4c) was consistent with the change in the trend graph of fastacting phosphorus (Fig. 3e). Sucrase activity was related to the metabolism of carbon (Bastida et al. 2016). At lower dilutions, the LP contained more carbon sources, which led to increased microbial metabolism. So the sucrase activity was higher (Fig. 4b). The activity of catalase was negatively correlated with pH. Therefore, a small dilution of LP with high organic acid content was more favorable to promote the activity of catalase (Fig. 4d) in the soil.
To further represent more visually the effects of pyrolysis temperature and dilution times on soil nutrients, physicochemical properties, and enzyme activity, we did a correlation analysis, as shown in Fig. 4e. The similarity of the color level indicates that the effect was close. As can be seen from the figure, the dark red color is mainly concentrated in the area of dilution multiples 50 and 400. Low dilutions have a positive effect on organic matter, total salinity, alkaline nitrogen, sucrase, and catalase activity in the soil. Higher dilutions had positive effects on fast-acting phosphorus, fastacting potassium, plant growth, alkaline phosphatase, and urease in the soil. In order to investigate more clearly which microorganisms are affected by LP at high and low dilutions, high throughput sequencing was performed in this study.

Effects of LP on soil microbial communities
We made gene sequencing on 16SRNA to evaluate the richness and diversity under various suitable conditions. Figure 5a displayed the Shannon-Wiener curve of bacterial communities in this research. As the increase of Effects of LP on sucrase activity in saline-alkali soils. c Effects of LP on alkaline phosphatase activity in saline-alkali soils. d Effects of LP on catalase activity in saline-alkali soils. e Correlation analysis of soil physicochemical properties, nutrients, and enzymatic activities with pyrolysis temperature and LP dilution multiplier measured sequence, the Shannon-Wiener curve leveled off. Generally, The Shannon and Simpson indices reflected the diversity and uniformity of samples, and Chao 1 and ACE reflected the richness of communities (Pd et al. 2021). As can be seen from Table 2, the Shannon values of samples were all higher than CK, indicating that the addition of LP was beneficial to enriching the microbial community. Simpson value in low dilution ratio samples decreased, while that in high dilution ratio samples increased. This was because LP at low dilutions contained more phenolics, which affected the biological activity of some microorganisms. And the appropriate concentration of LP had a positive effect on the growth of microorganisms (Mattana et al. 2019). Compared with the values of Chao 1 and ACE, CK increased, indicating that the richness of microorganisms in the samples was raised. It can also be seen from the abundance class curves (Fig. 5b). PCA results of multiple samples (Fig. 5c) showed that Multiple studies have shown, that at the phylum level, Proteobacteria had an absolute advantage in the alkalisaline soils (Bai et al. 2019). As shown in Fig. 5d, there are also distributions of Acidobacteria, Gemmatimonadetes, Chloroflexi, Firmicutes, and Acidobacteria. When LP (L-50) diluted 50 times was added, the abundance of Proteobacteria raised to varying degrees, while the abundance of Acidobacteria reduced and the abundance of Firmicutes greatly increased. The reason was that LP contains more (NH 4 ) 2 SO 4 at a lower dilution ratio, resulting in higher overall salt content than CK. The abundance of Proteobacteria, as the dominant bacteria, increased. From the analysis of order-level (Fig. 5e), Clostridiales had strong resistance under high salt conditions and became the dominant bacteria under the condition of abundant organic matter . From the analysis of genus level (Fig. 5f), it showed that Pseudomonas mostly appeared in a poor environment and releases catalase while consuming organic matter in the soil. This was also one of the reasons for the high catalase content in soil at a low dilution ratio (Fig. 4d). Soil organic matter content was too high, causing C: N to be higher than 70:1. Under this condition, nitrogen-fixing bacteria start to fix nitrogen, which was the main reason why Azoarcus became the dominant genus (Du et al. 2021). Therefore, the content of alkali-hydrolyzed nitrogen was higher at low dilution ratios. Desulfosporosinus (Pester et al. 2012), a reductive microorganism often found in environments of high sulfate concentrations, restored sulfate to stable sulfides. At a low dilution ratio, the abundance of dominant bacteria in the saline-soil environment increased and the diversity of bacteria was low due to the addition of sulfate. Soil fertility was poor which led to poor growth, but it also improved to a certain extent compared with CK. LP diluted 400 times added, the richness of Proteobacteria slightly reduced, while Acidobacteria and Gemmatimonadetes significantly increased, sequenced in phylum level. Studies have shown that Actinomycetes mainly undertook the decomposition of organic matter, played a beneficial role in improving soil fertility, and could provide certain protection against abiotic stress (Shi et al. 2022). The increase in the abundance of Gemmatimonadetes was attributed to the raising of available potassium, available phosphorus, and organic matter content (Koblížek et al. 2020). From the analysis of order-level, Micrococales and Rhizobiales (Aliashkevich et al. 2021) were the main phospho-solubilizing bacteria orders, and the abundance of these two orders was higher when the dilution ratio was higher, which was also one of the reasons why the content of available phosphorus in soil was higher. Nitrosomonadales became the dominant bacterium mainly because the soil was rich in NH4 + , under the action of which, NH4 + can be converted into nitrate that can be directly used by the Chinese Cabbage (Boden et al. 2017). From genus-level analysis, Sphingomonas and Pseudarthrobacter can be used for the degradation of aromatic compounds (Lha et al. 2016). Because LP contained part of phenolic substances, the emergence of these two bacteria also meant the degradation of phenolic substances. Therefore, under the high dilution ratios, Chinese Cabbage grew better.

Conclusions
Experimental results showed that the addition of LP increased the contents of soil organic matter and available nutrients. In particular, LP diluted 400 times at a pyrolysis temperature of 500℃ had a suitable concentration of organic acids. They could neutralize soil alkalinity and reduce soil salt stress through the effective complexation of salt ions. Additionally, acidic organic substances could compete with enzymes for adsorption sites on soil colloids and minerals, reduce enzyme adsorption and increase soil enzyme activity. The increase of the Actinomycetes ratio optimized soil population structure and improved soil quality. The positive amelioration effect of LP on saline soil significantly improved the growth index of Chinese cabbage.