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Effects of Biodegradation of Corn-Starch–Sodium-Alginate-Based Liquid Mulch Film on Soil Microbial Functions

In response to the problems of the poor degradability and mechanical properties of liquid mulch, natural non-toxic polymer compound corn starch and sodium alginate were used to prepare fully biodegradable liquid mulch. The preparation conditions of the mulch were optimized, and the mechanical properties of the mulch and the changes in the microbial community in soil with the mulch degradation were analyzed. The corn-starch–sodium-alginate-based liquid mulch film had an optimum performance at a tensile strength of 0.145 MPa and an elongation at a break of 16.05%, which was attained by adding 33.33% sodium alginate, 50% glycerol 22 and 4% citric acid to corn starch after moist heat modification. Fourier transform infrared spectroscopy analysis showed that the -COOH in sodium alginate could interact with the -OH in starch and glycerol through hydrogen bonding, thus, resulting in a denser structure and better mechanical properties of the liquid mulch as a non-crystalline material. The soil burial degradation study of mulch revealed that corn-starch–sodium-alginate-based liquid mulch degraded completely at 25 days macroscopically, and mulch degradation increased soil organic matter content. Microbial kinetic analysis showed that the abundance and diversity of the bacterial community decreased with the degradation of the mulch, which was conducive to the optimization of the bacterial community structure and function. Arthrobacter of the class Actinomycetes became the dominant microorganism, and its abundance increased by 16.48-times at 14 days of mulch degradation compared with that before degradation, and Acidophilus phylum (14 days) decreased by 99.33%. The abundance of fungal communities was elevated in relation to the main functional microorganisms involved in liquid mulch degradation, with Alternaria and Cladosporium of the Ascomycete phylum Zygomycetes being the most active at the early stage of mulch degradation (7 days), and the relative abundance of Blastocystis was significantly elevated at the late stage of mulch degradation (14 days), which increased by 13.32%. This study provides important support for the green and sustainable development of modern agriculture.

Introduction
Mulch films have been used for ground covering to retain heat and water, improve soil moisture, maintain soil structure, and prevent weeds and pests. However, the "white pollution" caused by the widespread application of mulch films is serious. The development of new environmentally friendly biodegradable mulching is of great significance to mitigating the greenhouse effect and surface pollution and ensuring food safety.
Biodegradable mulch is difficult to fully degrade [1] and needs careful laying in the field as it sometimes gets broken during soil application. As a new biodegradable soil cover material, the liquid mulch film could be more conducive to soil than traditional plastic films.
Soil was collected from the topsoil of a greenhouse in Shouguang City, Shandong Province, China. The soil properties in the top 20 cm were as follows: soil organic matter at 40.85 g/kg, pH at 8.1, electrical conductivity at 0.34 ds/m, and total dissolved salt at 6.68 g/kg.

Experimental Design 2.2.1. Preparation of Liquid Mulch Film
The modification of natural starch using hydrothermal treatment is an environmentally friendly physical modification method. It can rearrange the internal molecules of the starch granules [16] and partially pastes the starch granule. In addition, it will enhance the intermolecular interactions between starch molecules, improving the water resistance of starch and its resistance to enzymes.
It was concluded that a treatment humidity of 25%, a treatment temperature of 120 • C, and a treatment time of 8 h were the optimal hygrothermal frontal reaction conditions. The appropriate amount of hydrothermally modified corn starch was weighed in a beaker and stirred until the starch was completely pasted [17], dissolved sodium alginate [18] was added, and then citric acid and glycerol were added and stirred at 400 rpm/min for 2 h to obtain the liquid ground film. The liquid mulch film was formed by casting and the preparation process is shown in Figure 1.
This study will provide a theoretical basis and technical support for the application and promotion of degradable liquid mulch.
Soil was collected from the topsoil of a greenhouse in Shouguang City, Shandong Province, China. The soil properties in the top 20 cm were as follows: soil organic matter at 40.85 g/kg, pH at 8.1, electrical conductivity at 0.34 ds/m, and total dissolved salt at 6.68 g/kg.

Preparation of Liquid Mulch Film
The modification of natural starch using hydrothermal treatment is an environmentally friendly physical modification method. It can rearrange the internal molecules of the starch granules [16] and partially pastes the starch granule. In addition, it will enhance the intermolecular interactions between starch molecules, improving the water resistance of starch and its resistance to enzymes.
It was concluded that a treatment humidity of 25%, a treatment temperature of 120 °C, and a treatment time of 8 h were the optimal hygrothermal frontal reaction conditions. The appropriate amount of hydrothermally modified corn starch was weighed in a beaker and stirred until the starch was completely pasted [17], dissolved sodium alginate [18] was added, and then citric acid and glycerol were added and stirred at 400 rpm/min for 2 h to obtain the liquid ground film. The liquid mulch film was formed by casting and the preparation process is shown in Figure 1. The optimization of conditions for liquid mulch preparation was carried out by singlefactor experiments, based on the pre-experiment setting of 3 factors, each with 5 levels, and the reaction parameters were set as shown in Table 1. A degradability test was carried out after the liquid mulch film was formed by casting and was air-dried [19]. It was buried at a depth of 10 cm in the soil (pH 8.1, soil moisture 30%, temperature 25 • C). The mulch was removed and dried on days 0, 7, 14, and 21 to determine the weight loss rate of the mulch, and soil samples were collected using the five-point sampling method to determine the soil organic matter content and microbial community structure. Samples for microbial community structure determination were stored at −80 • C for backup and determined by high-throughput sequencing. Five-point sampling method was adopted for soil sampling. First determine the midpoint of the diagonal as the central sampling point and then select four points on the diagonal with the same distance from the central sampling point as the sampling point.

Analysis Methods
The mechanical properties of the mulch were determined using a microcomputercontrolled electronic universal testing machine (CMT4304, test speed 10 mm/min, sensor 50 N, Putian, China) [20,21].
The functional groups of the ground membrane were analyzed using a Fourier transform infrared spectrometer (Tensor II, Bruker, Germany) using KBr pellets at a mass ratio of 1:100 [22]. The geomembrane crystal structure was analyzed by an X-ray diffractometer produced by Rigaku corporation (Ultima IV, Tokyo, Japan) with a tube pressure of 40 kV, a tube current of 100 mA, a scan speed of 2 • /min, and a scan range of 2θ using Cu Ka radiation at a 2θ range from 5 • to 70 • with a scan step size of 0.02 • . X-ray energy spectrum (EDS) was used in conjunction with scanning electron microscope and transmission electron microscope to analyze the types and contents of elements in the micro area of materials.
The microbial analysis of soil community composition was determined by highthroughput sequencing. The bulk soils were collected from the place where the dry mulch film was buried and also kept in sterile plastic bags. The soil samples were immediately transported in an ice-packed container to the laboratory and stored in a cold room at 4 • C prior to analysis. Microbial community genomic DNA was extracted from soil samples and bacteria were amplified by forwarding primer 338F (5 -ACTCCTACGGGAGGCAGCAG-3 ) and reverse 806R (5 -GGACTACHVGGGTWTCTAAT-3 ) for the V3-V4 region of the 16S rRNA gene. The amplification of the fungus was performed in the ITS1F_ITS2R region by PCR reactions with the forward primer ITS1F (5 -CTTGGTCATTTAGAGGAAGTAA-3 ) and the reverse primer ITS2R (5 -GCTGCGTTCTTCATCGATGC-3 ). PCR reactions were repeated three times, and PCR products were extracted from 2% agarose gels, purified with the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Axygen, Union City, CA, USA), and quantified using a Quantus TM fluorometer (Promega, Madison, WI, USA). Alpha diversity index, colony structure, and function prediction analysis were performed on Meguiar's Bioinformatics Cloud platform (https://login.majorbio.com, accessed on 2 April 2021). Simpson indices were used to calculate species richness and relative abundance (alpha diversity), while Shannon indices were used to calculate species similarity (beta diversity) in different sampling units. Both of them can describe species richness and diversity. Chao indices were used to estimate the number of OTUs contained in a sample. The higher its value, the more species in the sample. Ace indices were used to evaluate the richness and evenness of species composition in the sample. The larger its value, the richer the species in the environment and the more uniform the distribution of species.

Calculation Method
The weight loss rate of the mulch was calculated according to Equation (1), and the corn-starch-sodium-alginate-based dry mulch was prepared as shown in Figure 1, where WL is the weight loss rate of the mulch, M 0 is the dry weight of the mulch before degradation (dried to constant weight at a constant temperature of 40 • C), and M 1 is the dry weight of the mulch after degradation [23].
(1)  Table 2 shows the main elements in the liquid mulch, as well as the mass ratio and atomic ratio of each main element. It can be seen that the liquid film is mainly composed of C and O elements, which are derived from each raw material for the synthesis of the liquid film. Among them, the content of element C is the highest, accounting for 58.09% of the total mass, and the number of atoms accounts for 64.94% of the whole, indicating that the liquid mulch is a carbon skeleton organic polymer. Element O is also an element constituting the liquid mulch film, and its mass and number of atoms account for 41.43% and 34.78% of the total, respectively. Na comes from sodium alginate with less content. In this study, the tensile strength and elongation at break of air-dried mulch were used as evaluation indices to optimize the conditions for the preparation process of liquid mulch, as the mechanical properties of liquid mulch are the critical constraints for its application. The effects of different additions of sodium alginate, glycerol, and citric acid on the tensile strength and elongation at the break of the mulch film are shown in Figure 2.

Results and Discussion
As shown in Figure 2a, the elongation at break and tensile strength of the mulch film exhibited a trend of increasing and then decreasing with the addition of sodium alginate. When the ratio of sodium alginate to starch was 1:3, the tensile strength reached a maximum value of 0.13 MPa, the elongation at break reached a maximum value of 14.19%, and the elongation at break and tensile strength decreased when changing the ratio of both. The improvement in mechanical properties is due to more hydrogen bonds formed between sodium alginate and starch as the content of sodium alginate increases, so the network structure of the polymer is more stable. The weaker mechanical properties may be attributed to the excess of sodium alginate and the decrease in starch content, as only a small amount of starch can combine with sodium alginate, Hence, the hydrogen bonding content between the two is reduced and the mechanical properties are lower.
As illustrated in Figure 2b, the elongation at break and tensile strength of the mulch film showed a trend of increasing and then decreasing with the addition of glycerol. The tensile strength reached a maximum value of 0.14 MPa and the elongation at break reached a maximum value of 15.02% when the mass ratio of sodium alginate to starch was 1:3 and the mass ratio of glycerol to modified starch was 0.7, similar to the test results when the mass ratio was 0.5. Therefore, the mass ratio of glycerol to starch was chosen to be 0.5 to maintain the optimized conditions, considering the production cost. Glycerol is a commonly used plasticizer that can improve the flexibility of the mulch material by As shown in Figure 2a, the elongation at break and tensile strength of the mulch film exhibited a trend of increasing and then decreasing with the addition of sodium alginate. When the ratio of sodium alginate to starch was 1:3, the tensile strength reached a maximum value of 0.13 MPa, the elongation at break reached a maximum value of 14.19%, and the elongation at break and tensile strength decreased when changing the ratio of both. The improvement in mechanical properties is due to more hydrogen bonds formed between sodium alginate and starch as the content of sodium alginate increases, so the network structure of the polymer is more stable. The weaker mechanical properties may be attributed to the excess of sodium alginate and the decrease in starch content, as only a small amount of starch can combine with sodium alginate, Hence, the hydrogen bonding content between the two is reduced and the mechanical properties are lower.
As illustrated in Figure 2b, the elongation at break and tensile strength of the mulch film showed a trend of increasing and then decreasing with the addition of glycerol. The tensile strength reached a maximum value of 0.14 MPa and the elongation at break reached a maximum value of 15.02% when the mass ratio of sodium alginate to starch was 1:3 and the mass ratio of glycerol to modified starch was 0.7, similar to the test results when the mass ratio was 0.5. Therefore, the mass ratio of glycerol to starch was chosen to be 0.5 to maintain the optimized conditions, considering the production cost. Glycerol is a commonly used plasticizer that can improve the flexibility of the mulch material by forming hydrogen bonds with starch molecules through hydroxyl groups, thus, changing the molecular structure of the starch [24].
As presented in Figure 2c, the tensile strength reached a maximum value of 0.16 MPa, and the elongation at break reached a maximum value of 16.05% when the mass ratios of sodium alginate to starch and glycerol to modified starch were 1:3 and 0.7, respectively, and the addition of citric acid was 4%. It showed that citric acid played a certain crosslinking role during the reaction process, which made the macromolecules combine effectively and improved the mechanical properties of the mulch film. The elongation at break and tensile strength of the mulch film was reduced when the amount of citric acid added was greater or less than 4%, owing to the combination of excess citric acid and starch, which led to increased difficulty in the intermolecular slip of starch, resulting in As presented in Figure 2c, the tensile strength reached a maximum value of 0.16 MPa, and the elongation at break reached a maximum value of 16.05% when the mass ratios of sodium alginate to starch and glycerol to modified starch were 1:3 and 0.7, respectively, and the addition of citric acid was 4%. It showed that citric acid played a certain cross-linking role during the reaction process, which made the macromolecules combine effectively and improved the mechanical properties of the mulch film. The elongation at break and tensile strength of the mulch film was reduced when the amount of citric acid added was greater or less than 4%, owing to the combination of excess citric acid and starch, which led to increased difficulty in the intermolecular slip of starch, resulting in lower elongation at break and tensile strength [25]. Citric acid is a tri-carboxylic acid used to cross-link polysaccharide molecules with each other. In addition, citric acid enhances the intermolecular interactions of liquid mulch and improves the performance of the mulch. However, the high content in citric acid and the consequent acid hydrolysis depolymerize the starch molecules into smaller polysaccharides and promote the destruction of the starch granules [26].
It was observed that the best mechanical properties of the liquid ground film were prepared by choosing 1:3 sodium alginate/modified starch (w/w), 1:2 glycerol/modified starch (w/w), and 4% citric acid addition.

Functional Group Analysis by FTIR
The functional group analysis was conducted on the solid starch and alginate films. The distribution of functional groups of liquid mulch and its components are shown in Figure 3a, which shows that liquid mulch is significantly different from modified starch and sodium alginate. Compared to modified starch and sodium alginate, the modified cornstarch-sodium-alginate-based liquid mulch has a stronger peak intensity at 3600-3000 cm −1 with reduced fine structure due to -OH stretching vibrational peaks in the corn starch and glycerol structures [27]. The presence of ester groups at 1720 cm −1 in the liquid mulch is created by combining citric acid with other polymers, indicating that crosslinks are formed. The carboxyl (-COOH) stretching vibration peaks of alginate appearing at 1640-1400 cm −1 (Wilpiszewska et al., 2019) and the absorption peaks between 1200 and 1000 cm −1 caused by the C-O stretching vibration of the glycosidic bond [28] are generated by the addition of sodium alginate. The -COOH in sodium alginate can interact with the -OH in starch and glycerol through hydrogen bonding to make the liquid ground film more compact and, thus, better serve to inhibit water evaporation and increase the effective temperature accumulation.
The distribution of functional groups of liquid mulch and its components are shown in Figure 3a, which shows that liquid mulch is significantly different from modified starch and sodium alginate. Compared to modified starch and sodium alginate, the modified corn-starch-sodium-alginate-based liquid mulch has a stronger peak intensity at 3600-3000 cm −1 with reduced fine structure due to -OH stretching vibrational peaks in the corn starch and glycerol structures [27]. The presence of ester groups at 1720 cm −1 in the liquid mulch is created by combining citric acid with other polymers, indicating that crosslinks are formed. The carboxyl (-COOH) stretching vibration peaks of alginate appearing at 1640-1400 cm −1 (Wilpiszewska et al., 2019) and the absorption peaks between 1200 and 1000 cm −1 caused by the C-O stretching vibration of the glycosidic bond [28] are generated by the addition of sodium alginate. The -COOH in sodium alginate can interact with the -OH in starch and glycerol through hydrogen bonding to make the liquid ground film more compact and, thus, better serve to inhibit water evaporation and increase the effective temperature accumulation.

Geomembrane Crystal Structure Analysis by XRD
The XRD spectra for the two main raw materials of corn-starch-sodium-alginatebased liquid mulch and prepared mulch were obtained for the solid starch and alginate films and are shown in Figure 3b. The characteristic crystalline diffraction peaks of corn starch after moist heat treatment were weakened and the crystallinity was reduced, but the crystalline structure remained. Sodium alginate showed two weak crystalline diffraction peaks at 32.90° and 34.06°, which proved that a small amount of crystalline

Geomembrane Crystal Structure Analysis by XRD
The XRD spectra for the two main raw materials of corn-starch-sodium-alginatebased liquid mulch and prepared mulch were obtained for the solid starch and alginate films and are shown in Figure 3b. The characteristic crystalline diffraction peaks of corn starch after moist heat treatment were weakened and the crystallinity was reduced, but the crystalline structure remained. Sodium alginate showed two weak crystalline diffraction peaks at 32.90 • and 34.06 • , which proved that a small amount of crystalline structure was also present in sodium alginate. The corn-starch-sodium-alginate-based liquid mulch only showed a broad and strong non-crystalline peak near 20.68 • , indicating that the crystal structures of modified corn starch and sodium alginate were gradually destroyed during the preparation of the liquid mulch and the mulch had obvious non-crystalline structures. Research has shown that the varied microstructure of the non-crystalline materials can be assigned to the irregular arrangement of particles. The non-crystalline structure characteristics gradually appear when the crystal structure is reduced, thus, enhancing the transparency, impact strength, and ductility of the mulch [29].

Degradation Performance of Corn-Starch-Sodium-Alginate-Based Liquid Mulch
The degradation rate of corn-starch-sodium-alginate-based liquid mulch film over time is shown in Figure 4a. The results showed that the mulch was gradually degraded with the extension of soil burial time. The mulch film weight was 20.12 g at 0 days, 12.63 g at 7 days, 7.21 g at 14 days, and 3.43 g at 21 days, the degradation rates were 37.23%, 64.17%, and 82.95% at 7 days, 14 days, and 21 days, respectively. Under the joint action of soil microorganisms and moisture, the mulch morphology changed from a large volume of dry mulch into small fragments; dry corn-starch-sodium-alginate-based mulch completely disappeared macroscopically on day 25. with the extension of soil burial time. The mulch film weight was 20.12 g at 0 days, 12.63 g at 7 days, 7.21 g at 14 days, and 3.43 g at 21 days, the degradation rates were 37.23%, 64.17%, and 82.95% at 7 days, 14 days, and 21 days, respectively. Under the joint action of soil microorganisms and moisture, the mulch morphology changed from a large volume of dry mulch into small fragments; dry corn-starch-sodium-alginate-based mulch completely disappeared macroscopically on day 25. As presented in Figure 4b, the soil organic matter of the control group without liquid mulch application was 40.85 g/kg, and the organic matter content increased by 2.06% and 3.40% at 7 days and 14 days of mulch application, respectively, compared to the control group. Pronk et al. [30] analyzed the relationship between the role of soil organic matter and microorganisms and showed that microorganisms are responsive to changes in organic matter. As an essential nutrient in the growth of microorganisms, small-molecule carbon in organic matter can effectively promote microbial reproduction and metabolism in soil.

Abundance and Diversity of Soil Microbial Communities
The Shannon and Simpson's indices showed in Table 3. The results indicated an overall decreasing trend with the degradation of liquid mulch, while the Ace and Chao indices showed different trends for bacteria and fungi.
The Shannon and Simpson indices indicated a gradual decrease in bacterial community diversity, with a 52.85% decrease in bacterial community diversity. The Ace As presented in Figure 4b, the soil organic matter of the control group without liquid mulch application was 40.85 g/kg, and the organic matter content increased by 2.06% and 3.40% at 7 days and 14 days of mulch application, respectively, compared to the control group. Pronk et al. [30] analyzed the relationship between the role of soil organic matter and microorganisms and showed that microorganisms are responsive to changes in organic matter. As an essential nutrient in the growth of microorganisms, small-molecule carbon in organic matter can effectively promote microbial reproduction and metabolism in soil.

Abundance and Diversity of Soil Microbial Communities
The Shannon and Simpson's indices showed in Table 3. The results indicated an overall decreasing trend with the degradation of liquid mulch, while the Ace and Chao indices showed different trends for bacteria and fungi. The Shannon and Simpson indices indicated a gradual decrease in bacterial community diversity, with a 52.85% decrease in bacterial community diversity. The Ace and Chao indices demonstrated that the bacterial community richness in soil decreased with the continuous degradation of liquid mulch and the bacterial community richness decreased by 48.89% after 7 days of degradation.
The Shannon and Simpson's indices in fungal community diversity had no significant change after 7 days of liquid mulch application compared to before. The Ace and Chao indices in fungal community richness showed an increasing trend. The fungal richness increased by 31.33% compared with before at 14 days of degradation.
There were 301,924 valid sequences after filtering and the coverage rate of all samples was higher than 99% in this study, which showed that the mulch degradation was conducive to the growth and reproduction of fungi and optimization of bacterial functions.

Community Succession of Soil Bacteria
The soil samples at 0, 7, and 14 days of mulch degradation were labeled as D_0, D_7, and D_14, respectively. As shown in Figure 5a, the dominant phyla observed in all treatment groups were Actinobacteria, Proteobacteria, and Acidobacteria. The relative abundance of Actinobacteria and Proteobacteria increased from 42.24% in the control group to 75.18% and 67.54% at days 7 and 14 of the mulch degradation, respectively. As suggested by a previous study [31], the heat treatment of maize starch with citric acid as a crosslinking agent resulted in resistant dextrins that promoted the growth of Actinobacteria, which are producers of compounds that are essential for human and animal health by decomposing soil organic matter, contributing to the global carbon cycle and increasing plant productivity [32]. The phylum Proteobacteria increased from 9.92% in the control to 23.05% and 30.68% at days 7 and 14, respectively. The increase in the abundance of the phylum Metamorphomonas also indicates an improvement in soil quality (Araujo et al., 2020). It colonizes high-nutrient environments rich in carbon and is complex trophic. The relative abundance of Acidobacteria phylum decreased from 10.50% in the control to 0.28% and 0.07% at 7 days and 14 days of mulch degradation, respectively. Acidobacteria is suitable for survival in acidic environments and low-nutrient soil conditions and can be used as an indicator of soil impoverishment [33]. Furthermore, Actinobacteria and Proteobacteria gradually replaced Acidobacteria as the dominant flora in the soil after mulching, indicating that the soil quality improved after mulching.  As presented in Figure 5b, the dominant soil bacterial communities were Actinobacteria, Alphaproteobacteria, and Gammaproteobacteria of the phylum Aspergillus at the phylum level. The most significant changes in Actinobacteria were observed at 7 days and 14 days after the application of liquid mulch, with the relative abundance of Actinobacteria increasing from 28.22% in the control to 74.64% and 67.30%, the relative abundance of Alphaproteobacteria increasing from 4.29% in the control to 7.95% and 10.62%, and the relative abundance of Gammaproteobacteria increasing from 5.63% to As presented in Figure 5b, the dominant soil bacterial communities were Actinobacteria, Alphaproteobacteria, and Gammaproteobacteria of the phylum Aspergillus at the phylum level. The most significant changes in Actinobacteria were observed at 7 days and 14 days after the application of liquid mulch, with the relative abundance of Actinobacteria increasing from 28.22% in the control to 74.64% and 67.30%, the relative abundance of Alphaproteobacteria increasing from 4.29% in the control to 7.95% and 10.62%, and the relative abundance of Gammaproteobacteria increasing from 5.63% to 15.10% and 20.07%, respectively. It was suggested that Alphaproteobacteria can promote the tolerance of graminaceous plants to elements, such as Co, Cr, and Ni in alkaline soils [34]. Furthermore, some bacteria in Gammaproteobacteria can use sulfur as the electron donor to fix CO 2 . The significant increase in the relative abundance of these three groups of bacteria indicated that the application of liquid mulch can promote crop growth and can facilitate carbon cycling in the soil.
As illustrated in Figure 5c, the most significant increase in the relative abundance is Arthrobacter of the class Actinomycetes after the application of liquid mulch, whose relative abundance increased from 2.78% in the control to 49.89% and 45.82% at 7 days and 14 days of mulch degradation, respectively. Studies have shown that Arthrobacter can degrade pesticides and heavy metal cadmium in the soil and promote plant rooting. In addition, the relative abundance of Nocardioides, Pseudarthrobacter, and Pseudoxanthomonas increased. Among them, Pseudoxanthomonas has the function of degrading cellulose [35]. Pseudarthrobacter can hydrolyze cellulose and is resistant to cold environments and solar radiation. The increased abundance of the above functional microorganisms promoted crop growth through improving soil carbon sequestration and toxicity reduction.

Community Succession of Soil Fungi
As shown in Figure 6a, the dominant fungi in the soil at different degradation times of liquid mulch were mainly Ascomycota, Basidiomycota, and Mortierellomycota. The relative abundance of Ascomycota, a saprophytic phylum that degrades wood, food, cloth, and leather in the soil and decomposes plant and animal residues [36], decreased by 3.81% and increased by 1.15% at 7 days and 14 days of mulch degradation compared to the control. The relative abundance of Basidiomycota first increased and then decreased with the degradation of the mulch film; it increased by 5.15% and 1.49% at 7 days and 14 days, respectively, compared with the control group. Basidiomycota helps plants obtain nutrients from the soil and plants produce glucose through photosynthesis to feed Basidiomycota in turn, which can also form symbiotic relationships with insects. The phylum Basidiomycota was found to exhibit significant in vitro degradation of hydrocarbons, such as polycyclic aromatic hydrocarbons, persistent organic pollutants, halogenated hydrocarbons, and aromatic hydrocarbons, phenols, explosives, and dyes [37].
As presented in Figure 6b, the dominant soil fungal communities included Dothideomycetes, Sordariomycetes, Saccharomycetes, Eurotiomucetes, Microbotryomyomycetes, and Microbotryomyomycetes, etc., at the phylum level. At 7 days and 14 days of mulch film degradation, the relative abundance of Dothideomycetes decreased by 10.05% and 11.66% compared with the control group, respectively. Most Dothideomycetes were plant pathogens, causing huge losses to cash crops [38]. The decrease in their relative abundance indicated that liquid mulching could help to control fungal diseases. Sordariomycetes are saprophytes that promote nutrient cycling in soil [39]. With the degradation of the mulch film, the relative abundance of Sordariomycetes first increased and then decreased by 28.72% and 19.82%, indicating that the fungus participated in the degradation process of the mulch film, and its abundance decreased with the degradation of the mulch film. The relative abundance of Saccharomycetes of Ascomycota increased gradually with the degradation of the plastic film and increased by 4.88% and 20.70% at 7 days and 14 days, respectively. Saccharomycetes can promote plant growth and reduce the arsenic content in the soil. Thus, it can be concluded that the degradation of the plastic film was beneficial in reducing the content of heavy metals and other harmful substances in soil and promoted crop growth.
As illustrated in Figure 6c, the dominant soil fungal communities were Alternaria, Cladosporium, and Gibberella at the genus level. The relative abundance of Alternaria of the ascomycetes was reduced by 16.41% and 14.29% at 7 days and 14 days of mulch degradation, respectively, compared to the control group. Alternaria are planted pathogenic bacteria that cause early blight and brown spot disease, which are particularly common in crops in arid regions [40], and the decrease in their relative abundance suggests that liquid mulch is effective in controlling crop diseases. The relative abundance of Cladosporium, a genus of ascomycetes, increased by 8.12% and 5.48% at 7 days and 14 days, respectively. Cladosporium is mainly distributed in soils with low salinity, and its relative abundance is positively correlated with the content of total nitrogen in the soil [41]. The application of liquid mulch may improve the saline soils, increase the content of nitrogen in the soil, and optimize soil quality. It was apparent that soil nitrogen fixation and disease resistance increased significantly with the degradation of the mulch. The abundance of Saccharomyces, Candida, and Penicillium in the class Saccharomycetales increased gradually with the degradation of the mulch film. by 3.81% and increased by 1.15% at 7 days and 14 days of mulch degradation compared to the control. The relative abundance of Basidiomycota first increased and then decreased with the degradation of the mulch film; it increased by 5.15% and 1.49% at 7 days and 14 days, respectively, compared with the control group. Basidiomycota helps plants obtain nutrients from the soil and plants produce glucose through photosynthesis to feed Basidiomycota in turn, which can also form symbiotic relationships with insects. The phylum Basidiomycota was found to exhibit significant in vitro degradation of hydrocarbons, such as polycyclic aromatic hydrocarbons, persistent organic pollutants, halogenated hydrocarbons, and aromatic hydrocarbons, phenols, explosives, and dyes [37]. As presented in Figure 6b, the dominant soil fungal communities included Dothideomycetes, Sordariomycetes, Saccharomycetes, Eurotiomucetes, Microbotryomyomycetes, and Microbotryomyomycetes, etc., at the phylum level. At 7 days and 14 days of mulch film degradation, the relative abundance of Dothideomycetes decreased by 10.05% and 11.66% compared with the control group, respectively. Most Dothideomycetes were plant pathogens, causing huge losses to cash crops [38]. The decrease in their relative abundance indicated that liquid mulching could help to control fungal diseases. Sordariomycetes are saprophytes that promote nutrient cycling in soil [39]. With the degradation of the mulch film, the relative abundance of Sordariomycetes first increased and then decreased by 28.72% and 19.82%, indicating that the fungus participated in the degradation process of the mulch film, and its abundance decreased with the degradation of the mulch film. The relative abundance of Saccharomycetes of Ascomycota increased gradually with the degradation of the plastic film and increased by 4.88% and 20.70% at 7 days and 14 days, respectively. Saccharomycetes can promote plant growth and reduce the arsenic content in the soil. Thus, it can be concluded that the degradation of the plastic film was beneficial in reducing the content of heavy metals and other harmful substances in soil and promoted crop growth.
As illustrated in Figure 6c, the dominant soil fungal communities were Alternaria, Cladosporium, and Gibberella at the genus level. The relative abundance of Alternaria of the ascomycetes was reduced by 16.41% and 14.29% at 7 days and 14 days of mulch

Biodegradation Mechanism of Corn-Starch-Sodium-Alginate-Based Liquid Mulch
The soil microbial community succession pattern during the degradation of cornstarch-sodium-alginate-based liquid mulch shows that fungi are the main microorganisms involved in the biodegradation of liquid mulch, and the bacterial community structure and function are optimized during the mulch degradation process. During mulch degradation, the diversity and abundance of bacterial communities in the soil gradually decreased and the abundance of dominant bacteria increased significantly. Arthrobacter of the class Actinomycetes was the dominant microorganism with the largest percentage of abundance, and its abundance increased by 16.48 times at 14 days of mulch degradation compared with that before degradation. The phylum Acidobacter (14 days) decreased by 99.33% compared with that before degradation. The abundance of fungal communities in the soil increased significantly with the degradation of the mulch, among which the Alternaria and Cladosporium of the Ascomycete phylum Ascomycota were the most active at the early stage of degradation (7 days). The relative abundance of Alternaria of the Ascomycete phylum decreased by 16.41% (7 d), and Cladosporium increased by 8.12% (7 days) compared with that before degradation. Nakazawa et al. [42] revealed that soft-rotting fungi of the Cysticercus phylum can secrete laccase as an oxidation key enzyme, while brown-rotting tannins are capable of chemical reactions without enzymatic involvement. Zheng et al. [43] found that the combined multivariate soil environment strongly influenced the composition and diversity of microbial communities, from which it can be inferred that changes in soil environment may be related to mulch degradation and metabolites produced by microbial growth and metabolic processes. Thus, the specific degradation mechanism needs to be investigated more thoroughly for degradation intermediates.
Related studies have shown that microorganisms of the phylum Aspergillus and Actinomycetes are important species involved in the degradation of some organic matter and polymeric materials [44,45], which is largely consistent with the dominant phylum species obtained from the above-mentioned community succession of soil bacteria. Huang et al. [46] stated that bacteria are mainly classified as autotrophic and heterotrophic, and there is a complex, non-specific network of interactions between bacterial functional groups.

Conclusions
The optimal process conditions for the preparation of corn-starch-sodium-alginatebased liquid mulch film involved adding 33.33% sodium alginate, 50% glycerol, and 4% citric acid to corn starch after moist heat modification. FTIR and XRD characterization showed that -OH and -COOH interacted with each other through hydrogen bonding to make the film structure denser, and the tensile strength of the film was 0.145 MPa, and elongation at break was 16.05%. The film was a non-crystalline material with good mechanical properties.
The corn-starch-sodium-alginate-based liquid mulch was completely degraded macroscopically at 25 days. Microbial kinetic analysis showed that the abundance and diversity of bacterial communities decreased and the abundance of fungal communities increased, and the application of liquid mulch favored the dominant microorganisms of Actinobacteria, Proteobacteria, Ascomycota, and Basidiomycota. The growth of oligotrophic Acidobacteria and some plant pathogens was inhibited. Hence, fungi were the main microorganisms involved in the biodegradation of corn-starch-sodium-alginate-based liquid mulch, and the application of liquid mulch was conducive to the optimization of bacterial community structure and function. The mulch degradation increased the soil organic matter content and the abundance of beneficial soil microorganisms and improved the soil disease and toxicity resistance.


s2fieldsofstudy:
Agricultural And Food Sciences
Biology

provenance: AgriculturalAndFoodSciences,2022-2022-Agricultural And Food Sciences-2022.jsonl.gz.gz

text:
Antibody Evaluation and Mutations of Antigenic Epitopes in the Spike Protein of the Porcine Epidemic Diarrhea Virus from Pig Farms with Repeated Intentional Exposure (Feedback)

The feedback strategy, or controlled exposure of pig herd to the porcine epidemic diarrhea virus (PEDV), significantly decreased losses during a severe outbreak in late 2013 in Taiwan. However, some pig farms still suffered from recurrent outbreaks. To evaluate the association between antibody titers and clinical manifestations, sera and colostra were analyzed from one pig farm that employed the feedback strategy. Furthermore, spike (S) gene full sequences from six positive samples of two farms with and without using feedback were compared to investigate the evolution of PEDV variants circulating in pig herds. The results in this study showed that high PEDV antibody titers do not correlate with the high rate of protection from PEDV infection. In addition, repeated feedback generated the emergence of PEDV variants with unique substitutions of N537S and Y561H in the COE domain and S769F in the SS6 epitopes. These mutations indicated the pathogenetic evolution of PEDV strains existing in the cycle of the feedback method. A very strict biosecurity practice to block the routes of pathogen transfer should be followed to achieve successful control of PEDV infections in pig herds.

Introduction
Porcine epidemic diarrhea (PED) is a highly transmissible disease that causes substantial economic losses to pork producers all over the world [1][2][3]. PED clinical signs include watery diarrhea, vomiting, dehydration, high morbidity, and mortality in the suckling piglets. The causative agent, porcine epidemic diarrhea virus (PEDV), is an enveloped, single-stranded, positive-sense RNA virus, belonging to the family Coronaviridae, subfamily Coronavirinae, genus Alphacoronavirus. The genome of PEDV is approximately 28 kb in length and comprises 2 overlapping open reading frames (ORFs) 1a/1b encoding for 16 non-structural proteins (nsps), then a series of downstream ORFs encoding the 4 structural proteins, spike (S), envelope (E), membrane (M), and nucleocapsid (N), plus an accessory protein gene ORF3 [4]. The PEDV S protein is a homotrimeric membrane glycoprotein that is composed of two subunits, S1 and S2, mediating receptor binding and cell membrane fusion, respectively [5,6]. The essential roles of S protein in host-virus

Serology
The concentrations of IgG in serum or IgA in colostra were detected by using a commercial PEDV ELISA kit (GENTAUR, Kampenhout, Belgium) and AniGen PED IgA ELISA (BioNote Inc., Hwaseong-si, South Korea), respectively, and performed following the manufacturer's instructions.
The neutralizing antibody titers of PEDV in sera and colostra were evaluated following a previous study [18], with some modifications. The sera and milk of the sows were diluted in two-fold serial dilution in serum-free DMEM medium before mixing with an equal volume of 200 TCID 50 PEDV and incubated for 1 h at 37 • C, then transferred to 96-well plates containing confluent Vero cell monolayers. After 2 days, CPE was observed using an inverted microscope.

RNA Extraction and RT-PCR
Samples were identified as PEDV-positive by the conventional RT-PCR method, which targets the ORF3 gene, and confirmed again by quantitative reverse transcriptionpolymerase chain reaction (RT-qPCR), performed as previously described [9,19].
From the positive samples, RNA of the virus was extracted by the AxyPrep Body Fluid Viral DNA/RNA Miniprep Kit (Bioscience, Unioncity, CA, USA). The cDNA was immediately synthesized with random primers by the High-Capacity cDNA Reverse Transcription Kit (ThermoFisher, Carlsbad, CA, USA).

Sequencing of S Gene and Phylogenetic Analysis
Sequences encoding the S gene (4161 bp) were amplified by PCR using GDP-HiFi DNA polymerase (GeneDireX, Las Vegas, NV, USA), with specific primers designed based on the sequences of the PEDV reference (Table 2), following a previous study [20] under the following conditions of adjusted thermal cycles: initial denaturation at 95 • C for 5 min, 35 cycles of denaturation at 95 • C for 30 s, annealing at 60 • C for 30 s, extension at 72 • C for 4 min 30 s, and a final extension at 72 • C for 5 min. The amplified PCR products were purified by 1% agarose gel electrophoresis. The PCR product was excised from the gel then purified using FavorPrep GEL/PCR Extraction (Favorgen Biotech Corporation, Pingtung, Taiwan). The purified PCR product was then ligated into the pGEM T-Easy vector system (Promega, Fichburg, WI, USA). The identity of clones was confirmed by automated DNA sequencing (Genomics, New Taipei City, Taiwan).

Data Analysis and Statistics
All data of the SN titers in serum were log-transformed as the base of 2 to show potential linear associations. Data are expressed as mean ± SEM. A Student's t-test was performed to compare treatment means between groups, and the serum antibody titers of each sow before and after the feedback were analyzed by the paired t-test. The serum antibody levels in piglets were categorized at three levels: low (L: SN ≤ 8 or ELISA IgG ≤ 25, IgA ≤ 1.5), medium (M: 8 < SN ≤ 32, 25 < ELISA IgG ≤ 40, 1.5 < ELISA IgA ≤ 2.5), and high (H: SN ≥ 32, or ELISA IgG ≥ 40, IgA ≥ 2.5). The associations of the categorical variables with the subsequent incidence of diarrhea were analyzed using the χ 2 test. Differences in which p < 0.05 were considered statistically significant.

Lactogenic Immunity Increased after Intentional Exposure (Feedback)
To investigate the immune responses of sows after intentional exposure to PEDV, sera were collected from 15 sows before and after feedback in farm SL. Results showed that a significant (p < 0.05) increase in antibody titers was observed either examined by the SN test (3.7 ± 0.6 and 21.5 ± 5.8 for before and after feedback) or ELISA (20.1 ± 2.7 and 27.5 ± 5.0 for before and after feedback) ( Table 3). Sera were also collected from two offspring of each sow seven days after farrowing. The SN titers against PEDV of piglet serum (77.2 ± 16.5) were significantly (p < 0.05) higher than those of sow serum (21.5 ± 5.8), and with a moderate positive correlation (r = 0.42) between each other.

Lactogenic Immunity Showed No Association with the Protection of Piglets from PEDV in the Fields
To determine the associations between antibody titers and clinical manifestations, the antibody titer distributions in pigs with and without diarrhea were compared. Sera were collected from 2 piglets 2 days after birth from each of 16 and 15 sows in farms SL and RS, respectively. Colostra were also collected from 85 sows on the day of farrowing in the SL farm. Piglets with clinical signs of diarrhea caused by PEDV were confirmed by RT-PCR. Results showed that there was no association of the serum antibody levels with diarrhea in piglets either determined by SN (χ 2 p-values of 0.15 and 0.78 for farms SL and RS, respectively) or ELISA IgG (χ 2 p-values of 0.30 and 0.39 for farms SL and RS, respectively) ( Figure 1). There were also no associations of the IgA antibody titers in sow colostra with the protection of piglets from PEDV infection observed with χ 2 p-values of 0.40 for farm SL (Figure 2). ELISA 15 20.1 ± 2.7 27.5 ± 5.0 * Data are presented as mean ± SEM. a,b Means with different superscript letters in a row are significantly different (p < 0.05).

Lactogenic Immunity Showed No Association with the Protection of Piglets from PEDV in the Fields
To determine the associations between antibody titers and clinical manifestations, the antibody titer distributions in pigs with and without diarrhea were compared. Sera were collected from 2 piglets 2 days after birth from each of 16 and 15 sows in farms SL and RS, respectively. Colostra were also collected from 85 sows on the day of farrowing in the SL farm. Piglets with clinical signs of diarrhea caused by PEDV were confirmed by RT-PCR. Results showed that there was no association of the serum antibody levels with diarrhea in piglets either determined by SN (χ 2 p-values of 0.15 and 0.78 for farms SL and RS, respectively) or ELISA IgG (χ 2 p-values of 0.30 and 0.39 for farms SL and RS, respectively) ( Figure 1). There were also no associations of the IgA antibody titers in sow colostra with the protection of piglets from PEDV infection observed with χ 2 p-values of 0.40 for farm SL (Figure 2). Figure 1. Comparison of the distribution of serum neutralization and ELISA antibody titers in piglets with or without diarrhea in farms SL (a,b) and RS (c,d). Each point represents one litter and sera were collected from 2 piglets 2 days after birth from each sow. For the SL farm, n = 9 and 7 for litters with or without diarrhea, respectively, and for the RS farm, n = 10 and 5 for litters with or without diarrhea, respectively.

Figure 1.
Comparison of the distribution of serum neutralization and ELISA antibody titers in piglets with or without diarrhea in farms SL (a,b) and RS (c,d). Each point represents one litter and sera were collected from 2 piglets 2 days after birth from each sow. For the SL farm, n = 9 and 7 for litters with or without diarrhea, respectively, and for the RS farm, n = 10 and 5 for litters with or without diarrhea, respectively.

Phylogenetic and Genetic Analysis
The full-length S gene of PEDV strains collected from SL and CL farms was analyzed to determine their phylogenetic and genetic relationship with 34 sequences of PEDV strains from GenBank (Table A1). The phylogenetic tree was divided into two major groups (G1 and G2) ( Figure 3). Phylogenetic analysis shows that the six strains in this study cluster within the G2b group, closely related to the Japanese strain (IBR-7/JPN/2014), but less related to other Asian strains, such as Chinese PEDV strains (CH/GSTS/2016), Vietnamese PEDV strains (HUA-PED45), and Korean PEDV strains (CNUP1F-2019). However, they differ genetically from European strains (including CV777). The six strains analyzed in this study form a different sub-group from the previous virus strains isolated in Taiwan

Phylogenetic and Genetic Analysis
The full-length S gene of PEDV strains collected from SL and CL farms was analyzed to determine their phylogenetic and genetic relationship with 34 sequences of PEDV strains from GenBank (Table A1). The phylogenetic tree was divided into two major groups (G1 and G2) ( Figure 3). Phylogenetic analysis shows that the six strains in this study cluster within the G2b group, closely related to the Japanese strain (IBR-7/JPN/2014), but less related to other Asian strains, such as Chinese PEDV strains (CH/GSTS/2016), Vietnamese PEDV strains (HUA-PED45), and Korean PEDV strains (CNUP1F-2019). However, they differ genetically from European strains (including CV777). The six strains analyzed in this study form a different sub-group from the previous virus strains isolated in Taiwan between 2013-2014 and 2016-2018. The full length of the S gene of all five strains from farm SL is 4161 bp (1386 aa). The nucleotide sequences are separated into two closely related lineages: Group A (PT01, 02, 05) and Group B (PT 03, 04). When aligning to the CV777 vaccine strain, the identification of five strains had several point mutations, showing a low nucleotide sequence identity at 93.39% to 93.63%.
In comparison with the previous Taiwan PED strains circulating between 2013-2014 and 2016-2018, the five strains collected in our study shared between 99.15% to 99.61% nucleotide sequence identities.  Bootstrapping with 1000 replicates was performed to determine the percentage reliability for each internal node. Horizontal branch lengths were correlated to genetic distances among PEDV strains, with different genogroups labeled on the right. Black solid circles represent PEDV strains isolated from the SL farm that used feedback. The hollow square represents CY4 strains isolated from the CL farm with no feedback. The scale bar represents nucleotide substitutions per site.

Alignment of Antigenic Epitopes of S Protein
The S protein comprises a total of 1386 amino acids for all six PEDV strains obtained from SL and CL farms. In this study, we focused on analyzing the antigenic regions containing epitopes capable of inducing neutralizing antibodies against PEDV. The amino acid sequence corresponding to the COE domain and epitopes SS2, SS6, and 2C10, were located in the residues 502-641, 751-758, 767-774, and 1371-1377, respectively [21][22][23].
Corresponding to the phylogenetic analysis shown in Figure 3, the five PEDV strains collected from the SL farm can be categorized into two groups when analyzed using protein sequence alignment software. Complete identities in the S protein were observed amongst the PT01, PT02, and PT05 strains (Group A) and between the PT03 and PT04 strains (Group B). A total of 13 substitutions (P27S, A169D, A174T, S182H, M214T, L299F, S769F, P885S, A1054V, P1094L, R1104K, G1225S, and E1382K) in the S protein were observed when comparing the amino acid sequences of Group B PEDV strains with those of Group A. The two groups share 99.06% sequence identity. The Group A and Group B strains exhibit 98.2% and 98.7% identity with CY4, respectively.
Overall, the sequence alignment of the S proteins demonstrated that the Taiwanese topotype strain TW-Pingtung-41 (GenBank no. KP276251.1) and the six strains identified in this study share 92.22% to 93.01% of amino acid identity with the PEDV prototype CV777. Both CY4 and Group B strains exhibit 98.85% of amino acid identity with Taiwanese topotype strain TW-Pingtung-41, but Group A strain shares a slightly lower identity of 98.34%.
Multiple alignments of amino acid sequences of each antigenic region in S proteins among the identified field PEDV strains in this study and those of 35 Taiwanese strains published in GenBank were conducted ( Table 4). The amino acid sequence of the COE domain of CY4 shares 100% identity with that of strain TW-Pingtung-41, while both Group A and Group B strains share only 98.57%. As compared with CY4 and 35 Taiwanese PEDV strains, the 5 strains from SL farms were uniquely characterized by substitution mutations of N537S and Y561H in the COE domain. Amino acid variations at residues D509A, H524P/S, K566N, L603I, Y606H, E608D/A, G612V/A/D, and E636V in the COE region were occasionally identified among those of published Taiwanese PEDV strains. Another unique substitution (S769F) in epitope motif SS6 of Group B strains but not of Group A was also observed. No mutations were identified in epitopes SS2 and 2C10.

Discussion
Intentional exposure of sows and gilts to PEDV via feedback of homogenized intestines could offer protective maternal immunity to the most susceptible suckling piglets and thus shorten the outbreak period and minimize economic losses on pig farms. However, determined by the method of feedback, the farm types and sizes, the herd management systems, and biosecurity measures implemented, the intentional virus-exposure practices might result in a negative or positive impact on pig farms [24,25]. Therefore, our research at periodically used feedback farms aimed to: (i) evaluate the relationship between passive humoral immunity and the time showing clinical signs and (ii) investigate the evolution of the PEDV variants that circulated in this farm.
The lactogenic immunity to protect neonatal suckling piglets via colostrum and milk is critical in the prevention and control of PEDV infection [26]. Several studies support that exposure to infected PEDV tissues can induce a significant increase in specific IgA and IgG levels [17][18][19]. Our data from the SL farm are consistent with these reports. Results from the SN and ELISA IgA analyses in this study showed significantly higher antibody levels in sow sera after feedback. In the SL farm, sows were given oral exposure to PEDVinfected materials. This practice might provide mucosal immunization. The previous studies showed that boosting inactivated virus in previously exposed sows may increase protection in nursing piglets against PEDV and maternally derived PEDV-neutralizing activity. The specific memory B cells could develop subsequent intentional exposure [27,28]. Memory B cells might be critically related to the ability to induce and secrete mucosalspecific antibodies [29,30]. In our study, PEDV-specific antibodies (IgA and IgG) were detected in colostrum and milk, implying that memory B cells responsible for IgA and IgG synthesis existed at the intestinal mucosa and can respond to this PEDV stimulation. Colostrum/milk antibody levels associated with a protective immune response in piglets may be a more reliable diagnostic tool than systemic antibody responses for detecting vaccination efficacy [27,31].
In piglets, humoral immunity is important for protection against PED. In one recent study at a Midwestern United States sow farm, the sows were given oral immunization by feedback after an initial PEDV outbreak. Two months after this feedback approach, all weaned pigs in that farm tested negative for PEDV [32]. It has been shown that the immune response of pregnant sows vaccinated with the PEDV S1 protein provided lactogenic immunity to the piglets through colostrum and milk [33]. However, the correlation between the antibody levels and the time of showing clinical signs in piglets is not clearly described. Few researchers have considered IgA antibody responses as a correlate of protection [28,34,35]. The previous report has also demonstrated that anti-PEDV antibodies through intraperitoneal administration could contribute to the protection of piglets against PEDV infection and the passively transferred circulating antibodies partially improved the course of infection [36]. On the other hand, in our study, there was no association of the serum antibody levels with the diarrhea symptom either determined by the SN test or ELISA IgG. Our results also indicated no associations of the IgA antibody titers in sow colostrum with the protection of piglets from PEDV infection. Moderate or even severe PED occurred in piglets with high titers of maternal antibody in the SL pig farm, indicating that the viral loads in the barn environment might exceed the protection threshold provided by the passively transferred lactogenic immunity. Current PEDV intentional exposure is frequently ineffective, and it should be noted that: (i) there is no standard, optimized, feedback protocol, (ii) the potential of contamination of the feedback material with other infectious pathogens, (iii) mutations altering the antigenic nature of feedback viruses, and (iv) the possibility of continuous re-infection. Indeed, several studies in recent years have indicated that hazard to the herd arises from the re-infecting virus (using feedback or live vaccines) from reversion or recombination to virulence [17,37,38]. Therefore, the feedback protocols should only be applied in urgent explosive outbreaks to initiate and maintain a high lactogenic immunity. Disinfection and strict biosecurity measures should be thoroughly followed for a successful control and eradication of PEDV in pig herds.
PEDV phylogenetic investigation in Taiwan recently confirmed that the primary PEDV strain is related to Genogroup 2b, closely related to the strains USA/Texas39/2013 and Thailand P1915-NPF-01511A [14,20]. Although the strains isolated in this study are closely related with the US Genogroup 2b, they have greater similarity with the Japanese strain IBR7/JPN/2014 than with Chinese or earlier Taiwanese strains. Currently, very limited research has been conducted on the evolution of the PEDV circulating on the farm using feedback. While there have been multiple reports on the incidence and genetic diversity of the PED virus in Taiwan, no studies of the virus circulation in closed farms that used feedback regularly have been conducted. This research displayed several point mutations in the full spike gene of five virus strains-notably potential significant mutations of antigenic epitopes in the S gene. From our study, PEDV strains sequenced in that farm in 2020 have several mutations within the S gene compared to earlier Taiwanese strains (2013-2014 and 2016-2018). Phylogenetic analysis categorized the five PEDV strains identified from the SL farm into two groups, showing that two variants coexist in the pig population. The coexistence of different PEDV variants within pig herds or even individual pigs with high frequency has been reported [18,39]. The repeated feedback practices implemented in the SL farm might promote the emergence of new PEDV variants. The CY4 strain identified from the CL farm without frequent feedback exhibited 100% identity on the COE domain of S protein as compared with the topotype strain TW-Pingtung-41, but the five strains identified from the SL farm shared only 98.57%. These results revealed the fact that a complicated, constantly mutated, mixed genotype infection of PEDV might occur in pig herds utilizing a repeated feedback program.
The antigenic epitopes inducing neutralizing antibodies against PEDV infection in pigs are mainly located in the S glycoprotein. The mutations therefore more frequently occur in the S protein of PEDV [40][41][42]. Mutations in the S protein can lead to a variety of structural and functional consequences and contribute to the lack of efficacy of PEDV vaccines [42][43][44][45]. In this study, the two particular substitutions, N537S and Y561H, within the COE domain of the S protein could be significant in the pathogenetic nature of the PEDV strain isolated from the SL farm. It is interesting to note that lethal strains identified in China, South Korea, Japan, Taiwan, Canada, Mexico, and the United States all have common substitutions (-LQDGQVKI-to -SQSGQVKI-) within the SS6 epitope region of the S1 gene, which may play a role in PEDV virulence [19]. However, in our study, the unique substitution S→F (-SQSGQVKI-to -SQFGQVKI-) on the SS6 region of Group B strains could be an important mutation point to analyze the B cell epitopes on the S gene. Interestingly, no such above-mentioned mutations were found in PEDV strain CY4 identified from the CL farm without frequent feedback, and only 10 km distant from the SL farm during the same period. Furthermore, the SL farm was a tightly closed herd, and the replacement breeding stock came from only one source farm free from PEDV infection. The PEDV strains with substitution mutations would be unlikely to originate from other pig herds. The above information suggests that the amino acids at the positions of 537, 561, and 769 in the antigenic regions of S protein might be the most susceptible residues subject to substitution mutation in pig herds under immense immune pressure of the host and the high viral loads in the environments. Further examination of the molecular structure of these mutations should be carried out to understand the significance of such changes within the antigenic epitopes of PEDV in more detail. Similar to another report [46], the sequences of SS2 (aa 751-758) and 2C10 (aa 1371-1377) motifs were quite conserved, and no substitution in these motifs was found in this study.

Conclusions
In the case of PEDV infection, passively transferred maternal antibodies cannot provide full protection against the virus in piglets under field conditions with high viral loads. A very strict biosecurity program should be followed to achieve successful control of PEDV infections in pig herds with feedback practice. PEDV variants with unique substitution mutations of N537S and Y561H in the COE domain and S769F in SS6 epitopes emerged in pig herds under repeated intentional exposure of PEDV to sows and gilts.  Informed Consent Statement: Not applicable.

Data Availability Statement:
The data that support the findings of this study are available in the main manuscript of this article.

Acknowledgments:
We are grateful to Martin Ryan, School of Biology, University of St. Andrews, UK, for proofreading this paper, and our colleagues at RCAB for their excellent assistance.

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


s2fieldsofstudy:
Agricultural And Food Sciences
Materials Science

text:
Effect of Grain Moisture Content and Roller Mill Gap Size on Various Physical Properties of Yellow Dent Corn Flour

Corn has six main varieties grown globally for animal feed, human consumption, and commercial/industrial purposes. Flour is an end-product of corn dry-milling. Products that are derived from corn flour often show differences in physical, chemical, and pasting properties due to corn varietal differences, milling methods, differences in flour particle sizes, and drying temperatures. The study aimed to determine the effect of different moisture contents of yellow dent corn and roller gap sizes, on the physical properties of the resulting corn flour. The possibility to use the flour in the manufacturing of animal feed and ethanol production was also considered. Yellow dent corn grain with moisture contents (MC) of 8%, 14.0%, and 18%, and roller gap sizes (GAP) of 0.1016 mm, 0.2032 mm, and 0.3048 mm were used. The Witt corrugated roller mill equipment with rollers of 1/32 inches corrugations was used to produce fine grits and flours. The particle sizes obtained were between 0.54 mm and 0.75 mm which increased with an increase in MC and GAP. Grain with 8% MC and GAP of 0.1016 mm and 0.2032 mm, and grain with 14.0% MC and GAP of 0.1016 mm can produce flour of particle sizes good for swine feed. Grain with 8% MC and GAP of 0.3048 mm and grain with 14.0.0% MC and GAP of 0.1016 mm and 0.2032 mm can produce flour of particle sizes good for ruminant feed. Grain with 18% MC and GAP of 0.1016 mm, and grain with 14.0% MC and GAP of 0.2032 mm can produce flour of particle sizes good for poultry feed. GAP of 0.1016 mm and 8% MC can produce flour of particle sizes suitable for the ethanol production industry. Flour preparation should purposely be done based on grain MC and GAP. The geometric mean diameter of particle sizes ranged from 0.54 mm-0.75 mm, and the geometric mean diameter of particle sizes increased with increasing MC and GAP. Also, grains with 8% MC had the highest loss in flour, and the higher moisture of 18% significantly affected the red color of flour.

Dry milling is used to physically change kernels into their constituent endosperms, brans, and germs (Johnson, 1991). Flour is an end-product of corn dry-milling. Food products that are derived from flour show differences in physical, chemical, and pasting properties due to differences in corn varieties (Pan, Eckhoff, Paulsen & Litchfield, 1996;Nago, Akiossoe, Matencio & Mestres, 1997;Sandhu, Singh & Malhi, 2007), methods used in milling (Nghi & Narasimha, 1994;Martinez-Flores, Martinez, Figueroa & Gonza-lez, 1998), differences in particle sizes of the flour (Bolade, Adeyemi & Ogunsua, 2009), and effect of drying temperatures on grains (Hardacre & Clark, 2006). The benefits of smaller particle sizes are to improve digestion of feed by animals, improve binding ability between particles during pelleting, improve the mixing efficiency of ingredients in a compounded feed, increase particle homogeneity, and prevent ingredient separation (Stark, 2016). Biomass feedstocks flow and compressibility depends mostly on moisture content (MC) and particle size distribution. When MC in bulk powder material is increased, the cohesion and adhesion of particles due to the formation of interparticle bonds get increased (Plinke, Leith, Boundy & Loffler, 1995;Fitzpatrick, Iqbal, Delaney, Twomey & Keogh, 2004), and flowability of the feed processing decreases (Yan & Barbosa-Canovas, 1997). An increase in particle size of ground cereals when MC is low decreases the bulk and particle densities of flours (Mani, Tabil & Sokhansanj, 2004a). The particle size of corn is affected by the production system of the grain, and feed manufacturing process (grain cleaning and grinding equipment) either by using a hammer mill (screen size, tip speed, screen opening, air systems) or roller mill (corrugations, roll differentials, and gap settings) (Stark, 2016;Koch, 1996;Waldroup, 1997). In roller milling, smaller grains may escape grinding (Douglas, Sullivan, Bond, Struwe, Baier & Robeson, 1990), and particles produced are irregularly shaped (Koch, 1996). The particles produced from a hammer mill are spherical and uniformly shaped (Reece, Lott & Deaton, 1985).
There is a need to optimize the grinding process and to develop particle size specifications for specific purposes. The grinding should give uniform particle sizes, predictable particle size distribution, and the grinding equipment should produce enough flour to meet the feed or ethanol manufacturing processes. Changing the gap sizes of roller mill equipment and using a grain of yellow dent corn with different MC could impact the physical properties of the resulting flour. For this reason, the objective of this study was to determine the effects that the different moisture contents of the yellow dent corn, and the roller gap sizes have on the physical properties of the resulting corn flours. The possibility to use the resulting flours in the feed manufacturing and ethanol production industries was also considered.

Experimental Design
Flour was prepared from commercial yellow dent corn samples. The corn grains with moisture contents (MC) were adjusted to 8%, 14.0%, and 18%. The roller gap sizes (GAP) used were 0.1016 mm, 0.2032 mm, and 0.3048 mm. A completely randomized factorial design was used.

Milling of Grain
A Witt Corrugated-Roller Mill was used to fractionate the whole grain into grits using a roller of corrugations of 1 / 8 inches. The obtained grits were further milled using corrugations of 1 / 32 inches to produce the flours. The roller mill with corrugations of 1 / 32 inches, had its GAP adjusted to 0.1016 mm, 0.2032 mm, and 0.3048 mm to obtain different resulting flours.

Moisture Contents of Grain and Flour
The MC of grain was determined in triplicates using samples of 30 g at 103º C for 72 h, following ASAE S352.2 (ASAE, 2001). The flour MC was determined using 2 g at 130 o C for 1 h (AACC International, 2022).

Loss in Flour and Particle Sizes of Flour
The loss in flour was determined as a percentage of the initial mass of the grit obtained from using 1 / 8 inches corrugated roller. It is caused by the technical performance of milling machinery or incompetence operator. In determining the particle sizes of the flour, 100 g of the flour was passed through stacks of different sieve sizes by using the Tyler Ro-Tap sieve shaker (RX-29). ASAE S319.4 (2008) method was used in this analysis with US Sieve numbers between 10 -270. The amount of flour on each sieve was used to determine the geometric mean diameter (D gw ) and the geometric standard deviation (S gw ). The calculations were performed according to the equations described in ANSI/ASAE Standard S319.4. D gw and S gw of flour particles were calculated using following equations; Where W i is mass on the i th sieve (g), d i is the nominal sieve aperture size of the i th sieve (mm).

Color of Flour
A Minolta Chroma Meter was used to measure the colors of the flours, and measurements were taken in triplicate. The color scores of the flours were reported in terms of 3-dimensional color space based on the following rating scale: L* 100 = white, 0 = black; a* +/-= red/green; b* +/-= yellow/blue. The color values of a typical white flour, for example, were L* value +92.5 whiteness, a* value -2.4 green colors, b* value +6.9 yellow color.

Analysis of Data
The data were analyzed using ANOVA to determine if mean values were significantly different, and if so, then Tukey Kramer HSD was used (at 5%) to identify where the significant differences in means did occur.

Effect of the Corn Grain MC and GAP on the Geometric Mean Diameter (D gw ) of Flour Particles for Feed Production
The D gw increased with increasing grain MC, but the difference in GAP did not affect the D gw . The grain with 8% MC had the smallest D gw (0.59 mm), and the D gw was higher for the grain with 14.0% and 18% MC ( Table 1). The effect of changing GAP on D gw was only observed in the interactive effect with the grain MC. In Table 2, the interactive effect shows that irrespective of the GAP, grain with 18% MC had the highest D gw . The grain with 14.0% MC had the highest D gw when the GAP of 0.2032 mm and 0.3048 mm were used. The D gw decreased with corresponding decreases in the MC and GAP, for example, 8% MC had the lowest D gw using 0.1016 mm. The results indicate that D gw was dependent on grain MC and GAP. Depending on the preferences of the feed producers, the grain of suitable MC and GAP with recommended D gw are to be used. The dried corn (8%) had a brittle endosperm that made the grinding very smooth than the grain with high MC (14.0% and 18 %). The soaked water in the high moisture corn (14.0% and 18 %) might have hardened the starch, endosperm, and seed coat which resulted in rough and large-sized particles. It has been reported that hard seeds produce a relatively low percentage of fine particles after grinding . According to Stark (Stark, 2016), a higher GAP produces large-sized particles. Rose et al. 2001 reported that endosperm hardness influenced the milling outcome of wheat, thus, harder endosperms produced larger, irregularly shaped particles while soft endosperm produced small-sized particles. Since the 8% moisture corn was dry, the endosperm was brittle and soft, and therefore smoother particles were produced. Comparatively, the endosperms of the 14.0% and 18% MC grain were hard and resilient to break which resulted in large-sized particles.
The advantages of using the 8% MC grain are observable in the digestive system of livestock and industrial processing of feed which requires small-sized particles. Small-sized particles have the advantage of an increased number of particles and surface area per unit volume, and therefore accessibility to digestive enzymes also increases (Goodbrand et al., 2002). Small-sized particles also make feed formulation processing less complex (Koch, 1996;Mathew, Hoseney & Faubion, 1999;Carvalho & Ascheri, 1999). Preparing different types of feeds for livestock (ruminants), swine, and poultry require particular-sized particles. The benefits of particle size reduction are to improve feed digestion, improve binding ability between particles, improve the mixing efficiency of ingredients in a compounded feed, increase particle homogeneity, and prevent ingredient separation (Stark, 2016;Ying & Allen, 1998;Stark, Jones, Goodband & Kalivoda, 2010). Using flour prepared from the grain with 14.0% MC and the smallest GAP (0.1016 mm) can be advantageous in the feed industry because the flour had a smaller D gw (0.62 mm). This is because flours with high MC and small-sized particles are reported to have many advantages. Reports indicate that finely ground flour with high MC increased the rate and level of rumen fermentation and digestion (Ying & Allen, 1998;Lykos & Varga, 1995;Knowlton, Glenn & Erdman, 1998), and digestion in swine (Steinhart, Tokach & DeRouchey, 2012).
However, there should be a limit to particle size used, because small-sized particles could increase the risk of rumen acidosis in cattle, reduce the dry matter intake and rumen fermentation, and milk characteristics (Ying & Allen, 1998). MC and particle size distribution contribute significantly to the flow and compressibility properties of biomass feedstocks (Fitzpatrick et al., 2004;Yan & Barbosa-Canovas, 1997). Grain with low MC (8%) and a smaller GAP (0.1016 mm and 0.2032 mm) can increase dust problems, increase feed processing cost, and also increase the incidence of gastric ulcers in swine (Steinhart, Tokach & DeRouchey, 2012). The digestive tract of poultry (gizzard) is influenced by feed particle size during development (Nir & Ptichi, 2001;Nir, Shefet & Aaroni, 1994b;Cumming, 1994). Generally, broilers require diets having relatively large particle sizes. However, small-sized particles are important when preparing feed pellets for poultry (Amerah, Ravindran, Lentle & Thomas, 2007). Different animals have different feed particle size requirements. Swine's feed efficiency improves using small-sized particles (0.54 mm and 0.57 mm D gw ), and poultry's feed efficient increases using large-sized particles (0.71-0.75 mm D gw ) (Stark, 2016;Nir & Ptichi, 2001;Stark et al., 2010;Steinhart, Tokach & DeRouchey, 2012). Therefore feed producers should be aware of the grain MC and GAP used to obtain appropriate particle size formation.

Effect of Grain MC and GAP on Geometric Mean Diameter (D gw ) of Particles of Flour for Ethanol Production
Another industrial usage of yellow dent corn is in the ethanol production sector. The particle size in ethanol production is important as it directly relates to the amount of starch released. In this study, the D gw was in the range of 0.54 mm to 0.75 mm (Table 1). The grain with 8% MC generally had the smallest D gw than the grain with higher MC, but the different GAP did not change the D gw . The effect of GAP was observed only in the interactive effect (Table 2). Lamsal et al. (2011) reported D gw in the range of 1.18 mm and 1.27 mm using the GAP between 0.2032 mm and 0.508 mm, and corn grain with 15% MC. The results of this study differ from that of Lamsal et al. 2011 and could be due to varietal differences in the corn grain used. Rausch et al. 2005 reported in their survey of nine dry-grind ethanol plants in the upper Midwest United States, that D gw of ground corn was 0.94 mm. This value was above the D gw obtained in our current study. However, a recent study by Liu, 2009 showed the D gw of ground corn particles from six dry-grind ethanol plants in Iowa and South Dakota to range from 0.43 mm to 0.52 mm. Therefore, it would be appropriate to use grain with 8% MC and GAP 0.1016 mm and 0.2032 mm to produce flour of suitable size for ethanol production.
Using grain with 14.0% and 18% MC produced D gw above the average size (0.48 mm) used in most ethanol plants even at the lowest GAP used (0.1016 mm). Reports have also indicated that small-sized particles increase ethanol productivity (Kelsall & Lyons, 2003;Naidu, Singh, Johnston, Rausch & Tumbleson, 2007;Yeh, Huang & Chen, 2010;Lamsal, Wang & Johnson, 2011;Khullara, Dien, Rausch, Tumblesona & Singh, 2013). The finer the flour, the higher the enzyme kinetics and quantity of sugars produced, because of the increased surface area of the small-sized particles available to enzymes (Kelsall & Lyons, 2003). According to Khullar et al. 2013, using a 0.08 mm sieve screen produced a high percentage of ethanol conversion than using a 2 mm and 6 mm. Small-sized particles were reported to cause several problems during the ethanol production including higher power requirements and harder separation process in the decanter and thin stillage evaporator. Considering the short tempering process time, the grain with 8% MC was appropriate. Hurburgh et al. 2014 reported grain MC to be theoretically between 8% and 14.0%. The smallest GAP (0.1016 mm) can best be selected because the D gw obtained using the grain with 8% MC could have high soluble solid content in the thin stillage of the ethanol fermentation. However, for grain with the same MC, changing GAP may not have any statistically significant effect on D gw of the flour (Table 1) and probably ethanol production. This observation supports the findings of Lamsal et al. 2011; different GAP tested for a grain of the same MC did not give any significant difference in the value of the ethanol produced.   Table 3, the varied GAP did not affect the flour MC and loss in flour quantity, however, grain MC resulted in a loss in quantity of the flour. The quantity of the flour lost significantly depended on grain MC. Due to this, the highest loss was observed in the grain with the lowest MC (8%) but was minimal in the grain with 14.0% and 18% MC. The dried endosperm of grain when ground easily shatters to disperse clouds of smooth powdery flour, and therefore most of the resulting flour is escaped and was counted as a loss. This might have been the reason for the highest flour loss in grain with 8% MC compared to moist grain which prevents the shattering and dispersing of the endosperms. This result was illustrated in Figure 1.

Effect of Grain MC and GAP on Loss in the Flour, and the Flour MC
The flour MC depended on the grain MC. The grain with the 18% MC produced flour with a corresponding high MC of 22%. Although high MC can result in deterioration, but it also could increase cohesion and adhesion of particles by forming interparticle bonds in bulk powdered materials (Plinke et al., 1995;Fitzpatrick et al., 2004), and flowability is decreased (Yan & Barbosa-Canovas, 1997). Hence, to minimize the loss in flour quantity, the MC of grain should be less than 8%. Additionally, the high flour MC could cause deterioration and reduces the feed's shelf-life.

Effect of Grain MC, GAP, and their Interaction (MC*GAP) on the Flour Color
The type and form of animal feed preparation correlate to some flour properties including flour MC, particle size, color, etc. Color is an important factor in feed processing and can make the end product acceptable or unacceptable to the feed buyer. In Table 4, GAP, and grain MC and GAP interaction did not have any effect on the color of the flour (L*, a*, and b*). But grain MC only significantly affected a* (red) color of the flour. The only significant effect on the color of the flour (a*) was the grain with 18% MC. Thus, a* (red) color was significantly the same for the flours from the grain with 8% and 14.0% MC ( Table 5). The red color of corn lies in the hull (HAES, 1904). As grain absorbs water the red pigment becomes very noticeable. Hence, the flour from the grain with 18% MC had the highest red color compared to the flours from the 8% and 14.0% MC. Thus, the compound for the red coloration reacted with the high amount of water, making it very noticeable. The result shows that irrespective of the MC and GAP, the flours had similar white (L*) and yellow (b*) colors. White color is a property that correlates best with processing; however, the increased MC and GAP did not affect the white color of the flour. Since the MC and GAP did not affect the white (L*) color this is a better indication for feed or food preparation. Figure 2 (ab) illustrates the effect of grain moisture content and roller gap on flour color.

Conclusions
The obtained particle sizes ranged from 0.54 mm to 0.75 mm. There was a corresponding increase in particle sizes as grain MC and GAP increased. In preparing swine feed which generally requires particle size of flour of around 0.5 mm, using grain with 8% MC and GAP of 0.1016 mm and 0.2032 mm or grain with 14.0% MC and GAP of 0.1016 mm can produce flour of the required particle size. This is to improve feed efficiency and reduce daily grain intake. In preparing ruminants' feed which generally requires flour particle size of around 0.7 mm, using grain with 8% MC and GAP of 0.3048 mm, or grain with 14.0% MC and GAP of 0.1016 mm and 0.2032 mm can produce flour of the required particle size. Reportedly, this improves milk production, digestion, and reduces gastric ulcers. For poultry feed of particle size which is generally above 0.7 mm, using grain with 18% MC and GAP of 0.1016 mm or grain with 14.0% MC and GAP of 0.2032 mm can produce flour of the required particle size. These particle sizes have been reported to enhance egg production, digestion, and broiler developments. Using GAP of 0.1016 mm and grain with 8% MC can produce flour of particle sizes suitable for the ethanol production industry. The white and yellow colors of the resulting flour were not affected by the different levels of MC and GAP. The effect of the grain MC on the flour color was only significant for the red color in the 18% MC grain. Per the purpose of the flour preparation (feed or ethanol production), the grain MC and GAP can be carefully selected to obtain desirable particle sizes.