A method for directional regulation of rice field algae-bacteria biofilms through waste hydrothermal carbonization liquid products

Abstract

The present invention belongs to the field of directional regulation of rice field algae-fungus biofilms, and specifically relates to a method for directional regulation of rice field algae-fungus biofilms by hydrothermal carbonization of waste liquid phase products, including a process of preparing the waste hydrothermal carbonization liquid phase product, and adding it to a rice field system with algae-fungus biofilms in an appropriate amount and manner, and thereafter maintaining the overlying water depth of the rice field soil at 2-5 cm within 12 to 21 days. Compared with the prior art, the present invention has the significant advantages of being able to well regulate rice field algae-fungus biofilms during use, maintaining a suitable biomass of the rice field algae-fungus biofilms, as well as the diversity and richness of the bacterial and fungal microorganisms therein, which is beneficial to the stability of the rice field microbial community, and also helps to promote or maintain rice growth, reduce ammonia emissions, and promote or maintain rice yield during the subsequent rice field planting process.

Description

Technical Field

[0001] The invention belongs to the field of rice field algae-fungus biofilms, and particularly relates to a method for directionally regulating rice field algae-fungus biofilms through waste hydrothermal carbonization liquid phase products. Background Art

[0002] Hydrothermal carbonization (HTC) has been widely used to treat high-moisture raw materials such as animal manure. HTC is a thermochemical process that uses water as a medium at 150-375°C in a closed environment, using autogenous pressure to rapidly degrade biomacromolecules such as proteins, fats, and lignin in biomass, leading to decarboxylation, polymerization, and dehydration reactions. Compared with traditional high-temperature pyrolysis, hydrothermal carbonization requires lower temperatures and consumes less energy. Hydrothermal carbonization of biomass produces solid products (i.e., hydrochar) and liquid products (i.e., HTC-AP). Hydrochar has advantages such as a large specific surface area, rich surface functional groups, and high porosity. It can be used as a fuel, adsorbent, soil remediation agent, and improver, and has become a current research hotspot.

[0003] During the HTC process, about 20-50% of organic matter will enter the liquid phase product, mainly in the form of polysaccharides, amino acids and humic acid; water-soluble inorganic ions, such as NH4 + , K + etc. will also enter the liquid phase product. Therefore, HTC-AP has potential application value. Some studies have used the liquid phase product of hydrothermal carbonization to produce methane through anaerobic digestion, as a nutrient source for microalgae, or applied to farmland as liquid fertilizer, achieving its recycling. Other studies have shown that certain components contained in HTC-AP, such as phenols and their derivatives, furans, pyridine, etc., may be toxic and inhibitory to organisms. When Leng et al. used a 20-fold diluted liquid phase product to cultivate algae, they found that it had an inhibitory effect on algae growth.

[0004] The soil-water interface of rice paddies is a wetland ecosystem containing a unique algal-fungal biofilm system. Frequent exchanges of matter and energy, as well as elemental cycles, occur between the soil and water. These biofilms play a vital role in the biogeochemical cycle of nutrients. For example, they effectively fix nitrogen, potentially accumulating tens of kilograms per growing season. Furthermore, these biofilms serve as the final barrier to ammonia volatilization from rice paddies. By regulating these biofilms, the ecological function of rice paddies can be maintained.

[0005] The properties and functions of algal biofilms are related to their microbial composition, physical structure, organic matter source and content, hydraulic conditions, and pollutant levels. HTC-AP influences the biomass and microbial community structure of algal biofilms. Appropriate addition and method can regulate algal biofilms by maintaining a reasonable biomass and reshaping bacterial and fungal community structures. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for regulating rice field algae-fungus biofilm by hydrothermal carbonization of waste liquid products.

[0007] In order to achieve the above-mentioned object of the invention, the first aspect of the present invention adopts a technical solution: a method for directional regulation of rice field algae-fungus biofilm by hydrothermal carbonization of waste liquid phase products, characterized by comprising the following steps:

[0008] The pig manure and water are placed in a closed hydrothermal reactor, heated to 170°C to 270°C, and maintained for 40 to 90 minutes, wherein the mass volume ratio of the pig manure to the deionized water is 1:(9-11) g / mL; naturally cooled to room temperature, the reactor is opened, the hydrothermal reaction product is collected, and the liquid phase product is obtained by filtering. The liquid phase product is the liquid phase product of the waste hydrothermal carbonization;

[0009] The liquid phase product of the hydrothermal carbonization of waste is added to paddy field soil having a rice field algae-fungus biofilm, and the depth of the overlying water in the paddy field soil is maintained at 2-5 cm for 10 to 21 days to regulate the rice field biofilm; the amount of the liquid phase product of the hydrothermal carbonization of waste added is: the TN content of the liquid phase product added to each hectare (ha) of paddy field soil is converted to (2-11) kgN.

[0010] Furthermore, the waste hydrothermal carbonization liquid product is applied to the paddy soil once, 12-21 days before rice transplanting. The application of the waste hydrothermal carbonization liquid product, in a relatively small amount, 12-21 days before rice transplanting, is used to regulate algal biofilms in the paddy field.

[0011] In addition, during the entire rice planting period after rice transplanting, the depth of overlying water is maintained at 3-5 cm, and water management is the same as conventional practice. In fertilization management, the phosphorus and potassium fertilizer application rates are exactly the same as conventional practices, and the nitrogen fertilizer application rate needs to be the conventional nitrogen fertilizer application rate (for example, 180-240 kgN / ha) minus the total nitrogen contained in the liquid product. The liquid product replaces 1.5-10% (mass percentage) of conventional nitrogen fertilizer.

[0012] Preferably, the waste hydrothermal carbonization liquid phase product needs to be left standing for 10-45 days (to achieve aging) before use. The waste hydrothermal carbonization liquid phase product has a shelf life of 1 year after preparation (excluding the aforementioned standing time).

[0013] Preferably, the waste hydrothermal carbonization liquid product used should meet the following conditions: pH 3.4-7, EC 1-8 μs / cm, TN 0.1-11 g / L, TP 0-1.0 g / L, TOC 2-35 g / L. More preferably, it should meet the following conditions: pH 5-7, TN 0.3-1 g / L.

[0014] Heavy metal limits for liquid products: mercury (Hg) (as element) ≤ 5 mg / kg, arsenic (As) ≤ 10 mg / kg, cadmium (Cd) (as element) ≤ 10 mg / kg, lead (Pb) (as element) ≤ 50 mg / kg, chromium (Cr) (as element) ≤ 50 mg / kg.

[0015] The hydrothermal reaction process does not require pressurization, and pressure is automatically generated during the heating process.

[0016] The hydrothermal reaction product produced by the hydrothermal carbonization is filtered through a mesh to obtain a liquid product; the waste hydrothermal carbonization liquid product is irrigated into a rice field at a nitrogen concentration of 40 to 150 mg / L at one time, that is, the waste hydrothermal carbonization liquid product is used after standing for 10 to 45 days, and a certain amount of the liquid product is irrigated into the rice field along with water, which is equivalent to diluting the liquid product so that the nitrogen concentration of the diluted liquid product is 40 to 150 mg / L.

[0017] Preferably, the amount of the waste hydrothermal carbonization liquid phase product added is: the TN content of the liquid phase product added to each hectare (ha) of paddy field soil is (3-7) kg N (low amount).

[0018] A second aspect of the present invention is to provide the use of the aforementioned method or rice field algae biofilm in maintaining or increasing the microbial diversity and richness of the rice field algae biofilm, wherein the method maintains the diversity and richness of bacteria in the rice field algae biofilm, increases the diversity and richness of fungi in the rice field algae biofilm, and increases or maintains the diversity and richness of algae in the rice field algae biofilm;

[0019] And the application of the aforementioned method or rice field algae-bacteria biofilm in reducing the humification degree of dissolved organic matter (DOM) in rice field water, or in purifying the water quality of rice field water (rice field algae-bacteria biofilm can degrade organic matter in rice field water).

[0020] Although there are a small amount of phytotoxic substances in the liquid products, these substances are easy to decompose, soluble in water and volatile. The liquid products are returned to the field about 2 weeks before transplanting rice. The "natural decomposition after return to the field" is used to eliminate phytotoxicity and ensure its safety when returned to the field.

[0021] Compared with existing technologies, this invention offers significant advantages: through a low liquid product dosage, a reasonable treatment time, and specific preparation and control methods, the liquid product is applied once approximately two weeks before transplanting rice seedlings, allowing for controlled biofilm growth. This effectively regulates the rice field biofilm during use, ensuring the diversity and richness of microorganisms within the biofilm, contributing to the stability of the rice field microbial community and facilitating the purification of paddy field water by the algal biofilm. Biofilms have ecological functions in rice fields, such as purifying water. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Composition and structural properties of dissolved organic matter in the liquid phase products (HTC-AP) at different temperatures. (a) and (b) are Van Krevenlent plots of H / C and O / C of DOM in M180 and M260, respectively; (c) and (d) are comparisons of DBE and NOSC of DOM in M180 and M260, respectively (DBE represents the number of unsaturated double bonds and rings in the molecule; NOSC indicates the degree of carbon oxidation in DOM); (e) is the percentage of different aromatic groups in DOM in the liquid phase products (the Aromaticity Index (AI) is an indicator of the degree of aromaticity of the organic compound's molecular structure; AI>0.67 indicates a condensed aromatic structure; AI>0.5 indicates the presence of an aromatic structure in the molecule); (f) is the percentage of different oxygen atoms in the DOM molecules in the liquid phase products;

[0023] Figure 2 The relative proportions of DOM three-dimensional fluorescence area integration in M180 and M260 (Ⅰ: aromatic protein region (Tyrosine protein) Ex: 200-250nm, Em: 280-330nm; Ⅱ: aromatic protein region (Typtophan protein) Ex: 200-250nm, Em: 280-330nm; Ⅲ: fulvic acid region (Fulvicacid) Ex: 200-250nm, Em: 380-550nm; Ⅳ: soluble microbial product region (Soluble microbial product) Ex: 250-340nm, Em: 280-380nm; Ⅴ: humic acid region (Humic acid) Ex: 250-400nm, Em: 380-550nm);

[0024] Figure 3Relative abundance histograms of the top 10 abundant phyla of bacteria and fungi, (a), (b) and (d), (e), the rest were merged into Others; NMDS ordination plots of different samples of bacteria and fungi, (c) and (f) (Stress < 0.2, indicating that NMDS can accurately reflect the degree of difference between samples);

[0025] Figure 4 Correlation network diagrams of the physicochemical properties (TOC, TN, TK, TP, and phenolic acids) of microorganisms and liquid products; (a) and (b) are genus microbial network diagrams of bacteria and fungi, respectively (edges between nodes represent the correlation between the nodes they connect, edge width represents the magnitude of the correlation, node size represents the number of links connecting this node, and red and green edges represent positive and negative correlations, respectively);

[0026] Figure 5 Symbiotic network diagram at the bacterial microbial order level. The network working diagram is drawn by selecting the TOP 600OUT groups; (a) and (b) are the microbial network diagrams with the addition of M180 and M260; (c) and (d) are the top 9 abundant order-level microorganisms in (a) and (b), respectively; (e) is the proportion of positive and negative correlations between microorganisms; (f) is the total number of nodes and edges in the network (the edges between nodes represent the correlation between the nodes they connect, the nodes represent the number of connections to this node, and the red and green colors of the edges represent positive and negative correlations, respectively;

[0027] Symbiotic network diagram at the order level of fungal microorganisms; (g) and (h) are microbial network diagrams with the addition of M180 and M260; (i) and (j) are the top 8 abundant order-level microorganisms in (g) and (h), respectively; (k) is the proportion of positive and negative correlations between microorganisms; (l) is the total number of nodes and edges in the network (edges between nodes represent the correlation between the nodes they connect, nodes represent the number of connections connecting this node, and the red and green colors of edges represent positive and negative correlations, respectively);

[0028] Figure 6 (a) Fluorescence characteristics of the four components (C1-C4) identified from the PARAFAC model of aquatic DOM and (b) loading diagram; (c) Maximum fluorescence intensity (Fmax) of the four aquatic DOM components at 0 h (marked as Begin) and 288 h (marked as End) and (d) three indices (FI, HIX, and BIX); M180 represents HTC-AP at 180°C, and M260 represents HTC-AP at 260°C; L, M, and H represent low, medium, and high input concentrations of HTC-AP, respectively;

[0029] Figure 7Schematic diagram of regulating rice field biofilm by waste hydrothermal char liquid products. DETAILED DESCRIPTION

[0030] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0031] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. The materials, reagents, instruments, etc. used in the following examples are all commercially available unless otherwise specified.

[0032] hydrothermal carbonization

[0033] High-temperature heating was performed in a hydrothermal reactor (pressure was automatically generated during the high-temperature heating process), with a pig manure (i.e., sun-dried pig manure) and deionized water ratio of 1:10 (g / mL). Two reactors (each containing a pig manure:deionized water ratio of 1:10) were sealed and heated at 180°C and 260°C, respectively, for 1 hour. The mixture was then allowed to cool naturally to room temperature. The reactors were opened, and the hydrothermal reaction products (i.e., all substances in the hydrothermal reactor) were collected and filtered with a mesh to remove solid matter. The resulting liquid products (i.e., the liquid products of hydrothermal carbonization of waste) were then allowed to stand for 30 days before use, designated M180 (liquid products obtained by hydrothermal carbonization at 180°C) and M260 (liquid products obtained by hydrothermal carbonization at 260°C).

[0034] Algae culture and application of hydrothermal carbonization liquid phase product (HTC-AP)

[0035] Soil blocks containing algal biofilms from the paddy fields of Jiangsu Academy of Agricultural Sciences were inoculated in an artificial climate chamber (28±1°C, 12h light: 12h dark cycle, 2800Lux) to simulate natural temperature and light conditions. 5mL, 10mL, and 15mL of HTC-APs (M180 or M260) were diluted with DI water into flasks to prepare HTC-APs solutions. Then, 5mL of algal culture solution pre-cultured for 15 days and 50mL of diluted HTC-APs were added to each incubator, and 40g of air-dried soil was added. The air-dried soil had a pH of 7.7 and a TOC of 9.7g / kg. -1 , TN 1.1g kg -1 , TP 0.9g kg -1 , TK 16.2g kg -1 , 0.05AP g kg -1 The soil surface area of the culture system is 0.0049m 2. They were respectively designated as M180-L, M180-M, M180-H (M180 group), M260-L, M260-M, and M260-H (M260 group). An incubator containing 50 mL of deionized water was used as a control and designated as CK. The control group (CK) and each treatment (M180 group and M260 group) were replicated three times. All incubators were provided with holes for gas exchange and placed in an artificial climate chamber (28±1°C, 12:12h light: dark cycle, 2800Lux) to simulate natural temperature and light conditions. The depth of overlying water in the paddy soil was maintained at 2-3 cm. After 288 h of incubation (i.e. 12 days), surface water samples were collected for 3D-EEMs analysis, and algal biofilms growing on the soil surface were collected for high-throughput analysis.

[0036] The algae and fungi cultivation process is as follows: the soil blocks containing algae and fungi biofilm from the rice fields of Jiangsu Academy of Agricultural Sciences (the soil blocks must contain microalgae in the rice fields and must also contain bacteria, which refer to bacteria and / or fungi) are inoculated into BG11 culture medium and cultured in an artificial climate chamber (28±1°C, 12h light: 12h dark cycle, 2800Lux) for 15 days to obtain algae and fungi culture medium.

[0037] The purpose of culturing the algae culture solution separately here is that since the air-dried soil lacks rice field algae biofilm, the algae culture solution needs to be added to the air-dried soil to simulate the paddy field soil with rice field algae biofilm growing in the natural environment (with water).

[0038] High-throughput sequencing

[0039] The genomic DNA of the sample was extracted, and then the purity and concentration of the DNA were detected by agarose gel electrophoresis. An appropriate amount of sample was taken in a centrifuge tube and diluted to 1 ng / μl with sterile water. 16S and 18S were amplified by PCR (Bio-rad T100 gradient PCR instrument). Equal concentrations were mixed according to the concentration of the PCR product. After thorough mixing, the PCR product was purified by electrophoresis using 1×TAE 2% agarose gel. The sequence with the main band size between 400-450 bp was selected, and the target band was recovered by gel cutting. The product purification kit used was the GeneJET gel recovery kit from Thermo Scientific. The NEB from New England Biolabs was used. Ultra TM The DNA Library Prep Kit for Illumina was used to construct the library. The constructed library was quantified by Qubit and tested. If qualified, it was sequenced using NovaSeq.

[0040] Physicochemical properties of HTC-AP

[0041] The characteristics of the liquid phase product HTC-AP after hydrothermal treatment are shown in Table 1. The TOC, phenolic acid and nutrient content (N+P2O5+K2O) of M180 are higher than those of M260, while the pH and EC of M260 are higher than those of M180. The TOC concentration increased from 6.938 g·L after treatment at 180℃ to 1.377 g·L -1 After being reduced to 5.657 g·L at 260℃ -1 The TP content at 180℃ is 6.717mg·L -1 After treatment at 180°C, the pH was 5.84, and as the temperature increased to 260°C, the pH rose to 6.50. The phenolic acid content at 260°C was lower than that at 180°C. HTC-AP is rich in Ca and Na, with M180 and M260 contents of 274.59 and 727.98 mg / L and 261.16 and 748.33 mg / L, respectively. It also contains Pb, Ni, Mn, Mg, Al, Na, Zn, Cu, and B.

[0042] Table 1. Liquid product characteristics

[0043]

[0044] Note: Total organic carbon (TOC), total nitrogen (TN), total phosphorus (TP), total potassium (TK)

[0045]

[0046] Composition and structural properties of dissolved organic matter in HTC-AP at different temperatures

[0047] The content of unsaturated hydrocarbons and carbohydrates in M180 is higher than that in M260. The content of lipids, condensed aromatic structures, tannins, and small molecules in M260 is also higher than that in M180. Double-bond equivalents (DBE) represent the number of unsaturated double bonds and rings in a molecule; the nominal oxidation state of carbon (NOSC) indicates the degree of carbon oxidation in DOM; and the aromaticity index (AI) evaluates the degree of aromaticity in the molecular structure of organic matter. An AI greater than 0.67 indicates condensed aromatic structures; an AI greater than 0.5 indicates the presence of aromatic structures in the molecule. The broadband spectrum of DOM in HTC-AP is concentrated at 150-500 m / z. As the preparation temperature increases from 180°C to 260°C, the intensity at 200-300 m / z decreases, while the intensity at 100-200 m / z increases, indicating that the content of small molecular substances in HTC-AP is relatively increased, and high MW (Molecular Weights) organic matter is continuously converted into low MW, which is related to the degradation of large molecular organic matter and further decomposition of small molecules. Figure 1 The protein content in (a) and (b) did not change substantially at different preparation temperatures, and the content was 6.60%. HTC-AP had the highest lignin content, with M180 and M260 being 60.39% and 60.85% respectively. Figure 1 (a) and (b) in ). Figure 1 The content of unsaturated hydrocarbons and carbohydrates in (a) and (b) is relatively high at a lower preparation temperature. The unsaturated hydrocarbons and carbohydrates in M260 are 32.54% and 54.74% lower than those in M180, respectively. The tannin content in M260 (4.10%) is higher than that in M180 (3.50%). Figure 1 In (a) and (b), it was found that the content of lipid substances and condensed aromatic structure compounds in M260 increased relative to M180.

[0048] By plotting a comparison chart of DBE and NOSC, we can analyze the degree of unsaturation and hydrophilicity / hydrophobicity of DOM. Figure 1 In (c) and (d), DOM at both temperatures is mainly characterized by negative NOSC values, indicating lower polarity and higher hydrophobicity. Compared with M180, the hydrophobic molecules in DOM at M260 shift to hydrophilic molecules, and the increase in hydrophilicity promotes the entry of hydrophilic harmful compounds into the water phase. Figure 2(d) AI is used to estimate the fraction of aromatic structures. From the figure, we can see that most compounds are concentrated in aliphatic compounds (74.65% and 72.38%), aromatic compounds account for 18.12% and 19.12%, and condensed aromatic hydrocarbons account for 7.24% and 7.91%. From 180℃ to 260℃, the proportion of aliphatic compounds in the total organic compounds decreases, while the proportion of aromatic structure compounds increases, which is consistent with the Figure 1 (b) in the same. Figure 1 (f) shows that the CHO compounds in M180 are mainly O4-O6, while those in M260 are mainly O5-O7. The CHO compounds containing more O significantly increase, while those containing less O significantly decrease.

[0049] Figure 2 As can be seen from (a) and (b), the DOM excitation / emission (Ex / Em) peak of HTC-AP is located in the fifth region of Ex / Em (250-400) / (380-500) humic acid, indicating that its fluorescence characteristic peak is related to humic acid. By regional integration, the 3D-EEM spectrum is divided into 5 regions, as shown in Figure 2. Figure 2 As shown in (c), the fluorescence intensity of each area is calculated and Figure 2 (d) shows that the relative content of humic substances (Zones III + V) is higher than that of other components, indicating that HTC is a humic acid process involving the conversion of organic waste. The relative content of humic acid substances decreased by 11.6% with HTC temperature, indicating that the relative content of humic acid substances in HTC-AP decreases with increasing HTC temperature. Protein substances (Zones I + II + IV) decreased by 23.2% with increasing HTC temperature.

[0050] Impact of liquid products on water quality

[0051] The component Fmax extracted by PARAFAC was used to characterize the relative proportion of DOM in the water at each sampling point. The results at 0 and 288 h are as follows: Figure 6 As shown. The C1 component corresponds to the marine humus peak M and is closely related to biological activity. The C2 component is similar to the lignin and tannin components, corresponding to humus-like substances, and similar components are found in rivers and sewage. The C4 component is related to the presence of fulvic acid compounds in natural soils. The C3 component cannot find a similar component in openpowder. Since the fluorescence intensity of DOM mainly depends on the concentration of the component, the maximum fluorescence intensity (Fmax) of each component is used to quantify the component content. At 288h, the Fmax of the C1 to C3 components in the aquatic DOM decreased by 51.92% to 100% compared with the initial stage (0h). For the C4 component, the Fmax at 288h of the HTC-AP treatment was significantly higher than that of the initial treatment, with an increase of 81.40% to 100%.

[0052] The fluorescence index (FI) indicates the source of humic substances in DOM. When the FI is greater than 1.9, it indicates a distinct autogenous origin, with the water body's DOM primarily derived from its own microbial activity. When the FI is less than 1.4, DOM is primarily derived from exogenous inputs, with the water body's own contribution to productivity being relatively low. The autogenous index (BIX) reflects the relative contribution of autogenous DOM. A BIX value greater than 1.0 indicates a high degree of DOM degradation and a distinct autogenous component. Values between 0.6 and 0.8 indicate a low autogenous contribution. The humification index (HIX) can also reflect the degree of DOM humification to some extent. When the HIX is less than 1.5, it indicates a biological or aquatic bacterial origin. An HIX between 1.5 and 3 indicates weak humic characteristics and a significant recent autogenous origin. An HIX between 3 and 6 indicates strong humic characteristics and a weak recent autogenous origin. An HIX greater than 6 indicates strong humic characteristics and a significant terrestrial contribution. At the initial stage (0 h), the FI values for aquatic DOM were less than 1.4 for all treatments, indicating a terrestrial origin. According to the BIX index value, the spontaneous composition of aquatic DOM in the initial stage ranged from 0.99 to 1.20. Aquatic DOM with a HIX value lower than 1.5 may be caused by the humification process mediated by organisms or aquatic bacteria. In the late incubation period (288 h), the FI value of aquatic DOM was higher than that of the control, ranging from 1.62 to 2.08. In addition, the FI value of the M180 treatment was higher than that of the M260 treatment. The BIX values of the M180 and M260 treatments (ranging from 1.31 to 1.61) were higher than those of the CK (0.73) ( Figure 3 The HIX value range of HTC-AP treatment (0.61-0.71) was relatively low, lower than the HIX value of CK (0.84).

[0053] Microbial diversity

[0054] In the 16S rRNA high-throughput sequencing, 575,533 raw data (PE) were obtained from the 7 samples sequenced by Illumina NovaSeq, of which 354,488 were effective tags that could be used for subsequent analysis, with an effective data volume of 61.59%. In the 18S rDNA high-throughput sequencing, 656,490 raw data (PE) were obtained from the 7 samples sequenced by Illumina NovaSeq, of which 456,644 were effective tags that could be used for subsequent analysis, with an effective data volume of 79.55%. The number of bacterial community OTUs was CK>M180-L>M260-L>M180-M>M260-H>M260-M>M180-H, indicating that the abundance of bacterial microorganisms exposed to liquid products was slightly lower than that of the blank; the number of eukaryotic community OUTs was M180-L(678)>CK(575)>M180-M(535)>M260-M(531)>M260-L(518)>M180-H(508)>M260-H(377). From the dilution curves between groups, it can be seen that the number of OUTs of A and C was not much different and both were higher than that of B, indicating that the addition of lower temperature and low concentration of HTC-AP was beneficial to the development of eukaryotic microbial community structure, while high temperature and high concentration of HTC-AP were not conducive to the survival of eukaryotic community structure.

[0055] According to the species annotation results, the top 10 species with the largest abundance at each classification level in all experimental and control groups were selected to draw the species relative abundance column cumulative graph. Taking the species relative abundance column cumulative graph at the phylum level as an example, Figure 3 The relative abundance of species at the bacterial phylum level is shown in Figure 3In panels (a) and (b), the main species in the algae and fungi samples in this experimental system were Proteobacteria, Firmicutes, unidentified bacteria, Bacteroidota, and Myxococcota. Proteobacteria and Firmicutes were highly abundant in all treatments exposed to the liquid product, while in the control, they only accounted for 26.51% and 6.52% of the abundance, respectively. This suggests that the addition of the liquid product favored the growth and reproduction of Proteobacteria and Firmicutes. The main eukaryotic species in the fungi were Chlorophyta, Ciliophora, unidentified_Eukaryota, Cercozoa, (unidentified_Discoba), unidentified_Fungi, Diatomae, Chytridiomycota, Ascomycota, Cryptomycota, and others. Ciliophora and Chlorophyta were more abundant in the experimental group (8.48%-80.43%) and 2.98%-80.57%), respectively, and were the main dominant eukaryotic species in the experimental group ( Figure 3 (f) in the figure shows that the presence of HTC-AP promotes their growth.

[0056] The NMDS analysis results of the bacterial OTU level are as follows Figure 3 As shown in (c), M260-L and CK are located in the third quadrant, M180-L and M260-M are located in the third and first quadrants respectively; M180-M, M180-H and M260-H are located in the fourth quadrant. The results show that M260-L and the control have a high similarity. Figure 3 As can be seen in (d), the microbial community structures of the M180 and M260 groups are quite different, which is affected by the liquid phase products, resulting in a large difference in the microbial community structure. The NMDS analysis results of the fungal OTU level are as follows: Figure 3 As shown in (g), the difference between M260-L and CK is small, and it is located in the first quadrant. Figure 3In (d), M260-L and CK appear on the same branch in the cluster tree, indicating similar species structure and abundance. M180-L and M180-M are located in the second quadrant, M180-H in the third quadrant, and M260-M and M260-H in the fourth quadrant. The presence of M180-M and M180-H, and M260-M and M260-H on the same branch, respectively, indicates that the microbial community structures in samples spiked with high concentrations of M180 and M260 are similar.

[0057] At a similarity threshold of 97%, OTU division was performed according to QIIME software, and α-diversity analysis was performed based on the obtained OTUs. The alpha diversity results of the sample bacteria and fungi are shown in Table 2. In the bacterial microbial community, 9346 OUTs were obtained after OTU clustering. The number of species (OTU number) observed visually in all experimental and control groups were: CK (1676), M180-L (1494), M180-M (1306), M180-H (952), M260-L (1443), M260-M (1191), and M260-H (1284). The Shannon index is the total number of classifications in the sample and their proportion, and the Simpson index is a measure of the diversity and uniformity of species distribution within the community. Both can reflect the microbial community and species distribution. The higher the value, the higher the diversity of the microbial community in the sample and the more uniform the species distribution. As shown in Table 2, among the experimental groups, the Shannon index and Simpson index in the M180 group ranged from 6.416 to 7.549 and from 0.946 to 0.984, respectively, with average values of 7.135 and 0.965. In the M260 group, the Shannon index and Simpson index ranged from 6.553 to 7.136 and from 0.948 to 0.977, respectively, with average values of 6.934 and 0.959. These results indicate that the order of bacterial microbial community diversity among the experimental groups was M180 > M260. The Chao1 and ACE indices reflect species richness, inferring a theoretical richness from observed results. This richness is closer to the actual richness, as observed species richness is often lower than the actual richness. The ACE index was highest in CK, followed by M180-L, M260-L, M180-M, M260-M, M260-H, and M180-H. M180-H and M260-H had the lowest OTU counts, indicating a high number of microbial species and community abundance in CK. M180-H and M260-H had the lowest bacterial species and community abundance. The α index indicated that the liquid product did not significantly affect the microbial community structure but altered the overall microbial richness, which was highly dependent on the concentration of the liquid product.

[0058] In the fungal microbial community, the number of species (OTUs) observed visually in all experimental and control groups were: C0 (575), M180-L (678), M180-M (535), M180-H (508), M260-L (518), M260-M (531), and M260-H (377). Chao1 represents the total number of species contained in the estimated community sample, and ACE represents the number of OTUs in the estimated community. Both are commonly used indices in the field of ecology to estimate the species richness of sample microorganisms. Their values indicate that M180 and M260 do not have a significant effect on the species richness of eukaryotic organisms. As can be seen from Table 2, the two diversity indices (Shannon and Simpson) indicate that M180 and M260 have a strong influence on the physicochemical parameters of the eukaryotic microbial community structure, which may have different effects on the growth of fungi within the community.

[0059] Table 2. Alpha diversity statistics of bacteria and fungi in the samples

[0060]

[0061] Microbial Network

[0062] By calculating the Spearman correlation coefficient between the microbial genus and the components of the added liquid phase product, the data that meets the requirements are selected to draw the correlation network diagram. Figure 5 As shown in (a), the four environmental variables TOC, TN, TP and Phenolic acids are highly correlated with bacterial species, while Sedimentibacter, Methylophilus and Bacillus are highly correlated with environmental factors. Sedimentibacter and Bacillus are positively correlated with environmental factors, while Methylophilus is negatively correlated with environmental factors. In the fungal correlation network diagram ( Figure 5 (b)) found that environmental factors had a negative impact on the vast majority of fungal genera.

[0063] By establishing a collinearity network diagram, we can understand the role and status of individual microorganisms in the entire community. At the bacterial order level, the network diagram of M180 consists of 559 nodes and 11,753 edges, of which 60.76% of the species are in a coexistence relationship and 39.24% are in a repulsive relationship. The network diagram of M260 consists of 591 nodes and 13,798 edges, of which 52.67% of the species are in a coexistence relationship and 47.33% are in a repulsive relationship ( Figure 5 (e) and (f) in the figure). Figure 5 (c) and (d) show the top nine orders of abundant microorganisms in the network, namely Burkholderiales, Lachnospirales, Rhizobiales, Oscillospirale, Bacteriovoracales, Chitinophagales, Clostridiales, Cytophagale, and Sphingobacteriales.

[0064] At the Fungal Order level, a positive interaction was observed between the M180 and M260 clusters. The M180 network graph consisted of 614 nodes and 7575 edges, with 75.26% of the species coexisting and 24.74% of the species repelling. The M260 network graph consisted of 527 nodes and 4978 edges, with 73.97% of the species coexisting and 26.03% of the species repelling. Figure 5 (k) and (l) in the figure). Figure 5 Panels (i) and (j) show the top eight orders of abundant microorganisms in the network: unidentified Eukaryota, unidentified Bacillariophyceae, unidentified Cercozoa, unidentified Chlorophyceae, unidentified Spirotrichea, Glissomonadida, and Rhizophydiales.

[0065] Effect of temperature on dissolved organic matter

[0066] At low temperatures (180°C), the content of macromolecular substances is relatively high, while at high temperatures (260°C), the content of small molecular substances is relatively high. At high temperatures, the content of lipid substances and condensed aromatic compounds increases relative to low temperatures.

[0067] Figure 1 In (c) and (d), DOM at both temperatures is dominated by negative NOSC values, indicating low polarity and high hydrophobicity. When the liquid phase products were characterized by three-dimensional fluorescence and regional integration, it was found that humic acids dominated the DOM. From 180℃ to 260℃, the proportion of aliphatic compounds in the total organic compounds decreased, while the proportion of aromatic compounds increased, which is consistent with the Figure 1 (b) in the same.

[0068] Impact of liquid products on water quality

[0069] During the 0-288h period, the fluorescence intensities of components C1, C2, and C3, except for component C4, decreased significantly in the HTC-AP treatment. The fluorescence intensity of component C1 decreased from 2.555-54.013 to 0.059-22.094, and the fluorescence intensity of component C2 decreased from 1.051-47.487 to 0.056-3.666. Component C1, as a humus-like fluorophore, is closely related to biological activity. Component C2, whose composition is similar to lignin, can degrade wood fiber materials. In the relative histogram of bacteria ( Figure 4 ), and the relative abundance of Firmicutes was higher in the HTC-AP treatment. Therefore, the decrease in components C1 and C2 is speculated to be due to the consumption of humic substances by microorganisms during their activity. Component C4 is similar to soil fulvic acid compounds, which play an important role in maintaining and improving soil fertility and environmental detoxification. The fluorescence intensities of component C4 at the beginning and end of the CK treatment were 0.334 and 0.277, respectively, with little difference. However, the fluorescence intensity in the HTC-AP treatment increased from 0 to 7.722 to 18.339 to 49.827. Therefore, it is speculated that this fluorescence intensity is largely not derived from the soil in the experimental system, but likely from the degradation of organic matter in the HTC-AP treatment by microorganisms.

[0070] Effects of liquid products on microbial community composition and diversity

[0071] The liquid product produced during the hydrothermal carbonization process contains a large amount of nitrogen, potassium and a small amount of phosphorus, and has great potential as a liquid fertilizer. The liquid product can be used to replace the 1.5-10% (mass percentage) of conventional nitrogen fertilizer required for the entire rice planting process. Before transplanting rice, the liquid product is applied to the rice soil with its own rice field algae and fungus biofilm for 12-21 days, and then the rice seedlings are transplanted. The water and fertilizer management of rice is the same as conventional practices, only the nitrogen content of the liquid product is deducted when applying base fertilizer to rice, that is, the nitrogen fertilizer application in the base fertilizer is reduced).

[0072] During the algal and fungal culture, low, medium, and high concentrations of M180 and M260 were added, respectively, and cultured for 12 days. Within the bacterial population, Proteobacteria, Firmicutes, and Bacteroidetes were found to be more abundant in all treatments exposed to liquid products than in the control (CK) culture. Furthermore, in the relative bacterial histogram, higher liquid product concentrations correlated with higher Firmicutes abundance. Firmicutes have significant potential for digesting high-concentration, recalcitrant lignocellulosic materials and possess strong environmental adaptability, playing a crucial biological role in various ecosystems. Within the fungal populations supplemented with liquid products, Chlorophyta and Ciliophyta were found to be dominant species, playing a leading role in the community. Chlorophyta was found to be more abundant in M180 (42.9%) than in M260 (21.2%), with the highest percentage in M180-M (80.6%). Chlorophyta, a phytoplankton phylum, is a primary producer in lake ecosystems and an important indicator of trophic level, reflecting changes in community status.

[0073] Potential interactions in microbial communities under liquid products

[0074] The indirect effects of environmental factors were understood by including the main occurrence environment in the network construction, such as by representing it as a node in the network, its influence on other nodes can be examined. In the bacterial community, TOC, TN, TP and Phenolic acids were found to be significantly correlated with Comamonas, Bacillus, Methylophilus, Achromobacter and Sedimentibacter. Figure 4In (a), all other genera showed positive correlations, except for a negative correlation with Methylophilus. TK showed a significant negative correlation with Methylophilus and a significant positive correlation with Magnetospirillum, Bacillus, and Sedimentibacter. Comamonas, Azospirillum, Bacillus, and Methylophilus all appeared in the top 10 most abundant genera in relative abundance. Furthermore, the relative abundance of Comamonas was observed to be higher in the M180 and M260-added cultures than in CK, with the exception of M180-H, where it increased with the addition of M180 and M260. This is primarily due to Comamonas's inherent tolerance to phenolic compounds at 500 mg / L, while the initial phenolic acid content in M180-H was as high as 616 mg / L. This phenomenon confirms that phenolic acids affect Comamonas. The addition of the liquid product is beneficial to the survival of Azospirillum, which can promote plant growth by secreting plant hormones, which is beneficial to plant growth in the ecosystem. The liquid product will inhibit Methylophilus, and the higher the concentration, the stronger the inhibitory effect.

[0075] In the fungal community, it was found that among the 7 associated fungal genera, except for Vermamoeba and unidentifiedSpirotrichea, which were positively correlated with TK, the others were negatively correlated with the external environment. Among them, unidentified_Eukaryota, Paracercomonas, (heterotrophic flagellates) Reclinomonas, and (Boshi bacteria) Poteriospumella all appeared in the relative abundance of the top 10 abundant genera. Paracercomonas can remove the main nutrients (N, P) and inorganic cations (such as Ca) from the waste nutrient solution. 2+ Mg 2+ 、Fe 2+ Poteriospumella can improve wastewater physicochemical properties and has good bioremediation potential. Except for the relative abundance of Poteriospumella in M180-L, which was 4.67% and significantly higher than the CK (0.14%), all other treatments were lower than the CK. Both Paracercomonas and Poteriospumella have properties that improve wastewater treatments.

[0076] A network is a collection of objects (nodes) connected by interactions (edges, arcs, and links) and is used to represent and understand interactions in different fields. For example, in the study of microbial associations, nodes can be microbial taxa, operational units, OUTs, etc., and edges represent some real or inferred associations (positive or negative). In the present invention, it was found that in the M180 bacterial network, 12.62% Burkholderiales, 6.21% Bacteriovoracales, and 5.65% Rhizobiales were key species; in the M260 bacterial network, 12.77% Burkholderiales, 5.29% Lachnospirales, and 5.29% Rhizobiales were key groups ( Figure 5 (c) and (d) in the figure). Its key groups can directly or indirectly affect other species and are extremely important in the ecosystem. Although the last two key species of the two are different, they both belong to the Proteobacteria phylum. The Proteobacteria phylum has a high degree of metabolic diversity and plays an important role in the decomposition of organic matter, carbon and nitrogen cycles, etc., and can provide nutrients for the growth of other bacteria. The number of edges in the bacterial symbiotic network of M260 is higher than that of M180, indicating that the relationship between the bacteria in this community is closer. Based on the percentage of positive / negative correlation ( Figure 5 (e) in the results) showed that M180 was conducive to the coexistence of bacteria, and Proteobacteria accounted for half of the relative abundance, and Proteobacteria were all key species in the network. Among fungal microorganisms, unidentified Eukaryota (eukaryotes), unidentified Bacillariophyceae (diatoms), and unidentified Cercozoa were key groups for M180 and M260, of which unidentified Eukaryota had similar proportions. The unidentified Bacillariophyceae in M180 was 10% higher than that in M260, while the surface area / volume value of diatoms was lower, and the rich nutrient salt environment was suitable for its survival. The number of edges in the bacterial symbiotic network of M180 was higher than that of M180, indicating that the relationship between the fungi in this community was closer. The percentages of positive / negative correlations in the fungal symbiotic network diagram were similar ( Figure 5 (k) in the.

[0077] This study examined the physicochemical properties of M180 and M260, as well as the effects of HCT-AP addition on water quality and the correlation and network characteristics of the diversity, community structure, and environmental characteristics of rice field algal biofilms. It was found that M180 had higher TOC, phenolic acid, and nutrient content (N+P2O5+KO) than M260, while M260 had higher pH and EC than M180. M180 contained relatively high levels of carbohydrates (saccharides) and unsaturated hydrocarbons, while the relative content of lipids, condensed aromatic structures, and oxygen-rich compounds increased with increasing temperature.

[0078] HTC-APs can be absorbed by surrounding plants, with a low C1-C3 composite fluorescence intensity and a high C4 fluorescence intensity, which is beneficial to the production of fulvic acid.

[0079] High-throughput sequencing analysis showed that HCT-AP addition did not significantly alter bacterial species diversity (Simpson index) but reduced species richness (Chao1 and ACE). Among fungi, the addition of M180-L increased community diversity and richness, while high levels of HCT-AP decreased both. The physicochemical properties of HCT-AP were positively correlated with bacteria and negatively correlated with eukaryotes. Proteobacteria formed the core bacterial communities of both M180 and M260, with closer relationships between bacteria in the M180 community than in the M260 community. Among fungi, unidentified Eukaryota and unidentified Bacillariophyceae were key species in both M180 and M260, and the fungi in the M180 community were also more closely related than those in the M260 community.

[0080] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any form, and any technical solution obtained by equivalent replacement or equivalent transformation falls within the scope of protection of the present invention.

Claims

1. A method for directional regulation of rice field algae-fungus biofilm by hydrothermal carbonization of waste liquid products, characterized in that: The steps include: Pig manure and deionized water are placed in a closed hydrothermal reactor, heated to 170°C to 270°C, and maintained for 40-90 minutes, wherein the mass volume ratio of pig manure to deionized water is 1:(9-11) g / mL; naturally cooled to room temperature, the reactor is opened, the hydrothermal reaction product is collected, and the liquid product is filtered to obtain the liquid product, which is the waste hydrothermal carbonization liquid product; The liquid phase product of the hydrothermal carbonization of waste was added to paddy soil with a rice algae-fungus biofilm. The depth of the overlying water in the paddy soil was maintained at 2-5 cm for 12-21 days to regulate the rice biofilm. The amount of the liquid phase product of the hydrothermal carbonization of waste added was: the TN content of the liquid phase product added to each hectare (ha) of paddy soil was converted to (2-11) kg N.

2. The method for directional regulation of rice field algae-fungus biofilm by hydrothermal carbonization of waste liquid products according to claim 1, characterized in that: The liquid phase product of the waste hydrothermal carbonization is applied to the paddy field soil once 12-21 days before rice transplanting.

3. The method for directional regulation of rice field algae-fungus biofilm by hydrothermal carbonization of waste liquid products according to claim 1, characterized in that: The waste hydrothermal carbonization liquid product needs to be left to stand for 10-45 days before use; the waste hydrothermal carbonization liquid product used must meet the following conditions: pH 3.4-7, EC 1-8 us / cm, TN 0.1-11 g / L, TP 0-1.0 g / L, TOC 2-35 g / L.

4. The method for directional regulation of rice field algae-fungus biofilm by hydrothermal carbonization of waste liquid products according to claim 1, characterized in that: The hydrothermal reaction process does not require pressurization, and pressure is automatically generated during the heating process.

5. The method of directional regulation of rice field algae-fungus biofilm by hydrothermal carbonization of waste liquid products according to claim 1, characterized in that: The hydrothermal reaction product generated by the hydrothermal carbonization is filtered through a mesh to obtain a liquid product; The liquid phase product of the waste hydrothermal carbonization is irrigated into the rice field at a nitrogen concentration of 40 to 150 mg / L.

6. The method of claim 1, wherein: The amount of liquid phase product added from the hydrothermal carbonization of waste is: the TN content of the liquid phase product added to each hectare (ha) of paddy soil is (3-7) kg N.

7. The rice field algae-fungus biofilm obtained by the method according to any one of claims 1 to 6.

8. Use of the method according to any one of claims 1 to 6 for maintaining or increasing the microbial diversity and richness of algal biofilms in rice fields, characterized in that: The method maintains the diversity and richness of rice field bacteria, increases the diversity and richness of rice field fungi, and maintains or increases the diversity and richness of rice field algae.

9. Use of the method according to any one of claims 1 to 6 or the rice field algae-fungus biofilm according to claim 7 in reducing the degree of humification of dissolved organic matter in rice field water, or in purifying the water quality of rice field water.

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