Influence of organic liquid fertilizer developed from livestock manure on the growth, antioxidant activities, and soil microbial populations of Chinese cabbage

Influence of organic liquid fertilizer developed from livestock manure on the growth, antioxidant activities, and soil microbial populations of Chinese cabbage | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Influence of organic liquid fertilizer developed from livestock manure on the growth, antioxidant activities, and soil microbial populations of Chinese cabbage Junkyung Lee, Na-Yeon Jo, Su-Yeon Shim, Tran Yen Linh Le, Woo Yong Jeong, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2964376/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Effluents from livestock excretion have worldwide environmental disadvantages, such as air and water pollution. However, livestock manure and organic liquid fertilizers developed for the proper management of livestock excretions can be used as environmentally friendly fertilizers for sustainable agriculture. Therefore, we investigated the effects of organic liquid fertilizers on the growth and antioxidant accumulation in Chinese cabbage ( Brassica rapa subsp. Pekinensis ). Results Three liquid fertilizers, chemical liquid fertilizer (CLF), fermented liquid manure (FLM) from pig droppings, and liquid bio-fertilizer (LBF) from Chlorella cultured in purified organic liquid manure, were used in this experiment. Plant performance was observed and soil microbial changes caused by these liquid fertilizers were analyzed using 16S rRNA sequencing. We observed the highest plant growth in terms of plant length, chlorophyll concentration, width and length of leaves, and fresh and dry weights in the CLF group. LBF led to improved plant growth compared to FLM. Chlorophyll concentrations and color measurements of the plants were higher with LBF than with FLM. Interestingly, LBF affected plant substances with respect to sugar, ascorbic acid, and antioxidants in Chinese cabbage compared to CLF and FLM. The highest total polyphenol and flavonoid content, antioxidant activity, nitrite-scavenging capacity, and reducing power were observed in the LBF group. Significant changes in the bacterial population were observed in amplicon sequence variant analysis; the presence of Verrucomicrobia increased in soils after FLM and LBF treatments. LBF-treated soils had a higher abundance of Proteobacteria than FLM-treated soils. Conclusions We studied the effects of organic fertilizers across different liquid fertilizers on plant growth, antioxidants, and soil microorganisms. Especially, the LBF as organic bio-liquid fertilizer improved the plant growth and substances in Chinese cabbage under a controlled environment agriculture system. We have identified the specific bacterial species regarding the production process of liquid fertilizers in the change of soil microbial communities by the short-term experimental treatment. Thus, the proper development of organic bio-liquid fertilizer can contribute to the organic waste-recycling systems of livestock excretions for sustainable agriculture. organic fertilizer bacterial community bio-liquid fertilizer Chinese cabbage antioxidants Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1 Introduction Chemical fertilizers have played an important role in maintaining sufficient crop production owing to the increased food demand of a growing human population, despite decreasing farmland area [ 1 , 2 ]. However, the excessive use of chemical fertilizers can cause environmental pollution. For example, 50% of the nitrogen and 90% phosphorus of applied fertilizer in the cultivation region are released into the atmosphere or water sources. These released materials lead to the formation of greenhouse gases in the atmosphere, saline contamination in soil, and the eutrophication in the aquatic ecosystems [ 3 , 4 , 5 ]. Diverse organisms, such as soil microbes, humans, plants, and animals, in natural ecosystems have been adversely affected by the imbalance of soil nutrient ratios due to the use of chemical fertilizers [ 6 , 7 ]. Therefore, environmentally friendly organic fertilizers have been suggested for sustainable agriculture [ 8 , 9 , 10 , 11 , 12 ]. The agricultural traits of kiwi plants ( Actinidia deliciosa ) were improved, with an increase in crop production and plant nutrition, using organic fertilizers with crop residues and other biological wastes [ 13 ]. Furthermore, the availability of organic manure improved the growth, yield, and mineral content of okra ( Abelmoschus esculentus ) in comparison with NPK 15-15-15 fertilizer [ 14 ]. In a previous study, we found that the growth of maize was enhanced by supplying a mix of Hanwoo (Korean cattle) manure and chemical fertilizer compared to chemical fertilizer alone [ 15 ]. Studies on organic fertilizers have provided useful information for the development of sustainable agriculture [ 16 ]. Microbiome soil inoculation, which can replace synthetic farm inputs, has positive effects on the soil environment and plant growth; many studies on soil microorganisms have been conducted in recent years [ 17 , 18 ]. Plant growth-promoting bacteria (PGPB) have direct and/or indirect effects on the solubilization of soil nutrients (P, K, and Fe) and enhance tolerance to pests and diseases [ 19 , 20 ]. For example, one type of PGPB, rhizobacteria, improves water and nutrient uptake and tolerance to abiotic and biotic stress in the interaction between the microbiome and the plant [ 21 ]. Bio-fertilizers, including Bacillus megaterium var. phosphaticum and Azotobacter chroococcum , increased the dry weight of the roots and shoots of maize compared to untreated plants [ 22 ]. Some studies have attempted to understand the combined effects of two or more microbes on crop cultivation; however, the exact mechanisms underlying the diverse biological effects of soil microorganisms remain unclear [ 19 , 23 ]. Chlorella is a photosynthetic microorganism with a high potential for the production of useful metabolites [ 24 ]. The hormones auxin and cytokinin, which regulate plant growth and development, are increased during the cultivation of Chlorella and interact with plant metabolism [ 25 , 26 ]. These results suggest that the use of bio-liquid fertilizers with microorganisms will be useful for the improvement of crop production and soil amendment. Here, we attempted to produce the organic bio-liquid fertilizer by Chlorella cultivation with media developed from livestock manure. The Chinese napa cabbage ( Brassica rapa subsp. pekinensis (Lour.) Hanelt) is the most popular vegetable in East Asia, China, and Korea and is used for making kimchi, a traditional Korean fermented food. Chinese cabbage is a highly adaptable plant that is grown extensively worldwide and contains many nutrients, including vitamin C, dietary flavonols, crude fiber, and carotenoids [ 27 , 28 ]. This study aimed to enhance the quantity and quality of greenhouse crops, through the application of liquid fertilizers. We observed the effects of different organic liquid fertilizers on plant growth, antibiotic activities of Chinese cabbage, and soil bacterial communities compared to chemical fertilizers. This study provides useful information for realizing environment-friendly agriculture in greenhouses. 2 Materials and Methods Plant growth and fertilizers Chinese napa cabbage ( Brassica rapa subsp. pekinensis (Lour.) Rupr.) seeds were obtained from the Danong Company (Gyeonggi-do, Korea). The seeds were grown in soil-filled pots (18 × 18 × 30 cm) in the greenhouse of the Agricultural Technology center (located in Hoengseong County, Gangwon-do, Republic of Korea) under an average temperature of 25℃ and an average humidity of 65%. Plants treatments were performed with three independent replicates of each experimental unit. NPK fertilizer (21-17-17) used as the chemical liquid fertilizer treatment (CLF) was obtained from the Namhae Chemical Corporation (Jeollanam-do, Korea). The fermented liquid manure (FLM) was created from livestock manure, which was provide from hog farm (37°30'33.1948211615924"N 128°04'56.5629151080066"E) after the livestock excretion had decomposed for one year. The organic medium developed from livestock manure was produced following a previously described method [ 29 ] and was used to grow Chlorella fusca , which was provided by the National Institute of Agricultural Sciences, Republic of Korea. Chlorella was grown in an incubator (Fig. S1 ) to a concentration of 10 7 cells/mL at a mean temperature of 28℃. The incubator was comprised of LED modules (FNB-240LED; F&B Nature, Chungju, Korea) with red and blue light (16 h/8 h day/night cycle) and an air supply providing 0.1 m 3 air/m 3 ·min [ 30 ]. The Chlorella -incubated medium was filtered using a tubular continuous centrifuge (J-1050A; Hanil Sci-Med, Chungcheongbuk-do, Korea) at 12, 000 × g to obtain the liquid bio-fertilizer (LBF). The liquid fertilizers were treated with 1.5 mS/cm electrical conductivity (EC). After sowing the seeds, liquid fertilizers were supplied twice per day (50 mL at both 9:00 a.m. and 6:00 p.m.) using a dropper. Survey of plant growth, chemical components, and antioxidants Plant growth (plant length, fresh and dry weight, leaf color and length, and chlorophyll content), chemical components of the soil and liquid fertilizer (pH, EC, total N, NH 4 + -N, NO 3 − -N, P, K, Na, Ca, and organic matter [OM]), and antioxidants (total polyphenol and flavonoid contents, 2,2-Diphenyl-1-picrylhydrazyl [DPPH] and nitrite scavenging activity, reducing power, and Ferric Reducing Antioxidant Power [FRAP]) were all analyzed using previously described methods [ 15 ], and the plant substances were extracted by methanol. The standard substances were quercetin for total flavonoid and gallic acid for total phenol, DPPH, and FRAP assay, and then the proper standard curves were estimated (Fig. S2 ). Leaves of Chinese cabbage grown for 56 days were used for antioxidant analysis and color measurement. Colors (L (lightness), a (redness), and b (yellowness)) were measured using a Hunter Lab Colorimeter (ND-300A; Nippon Denshoku, Tokyo, Japan). Soil microbiome analysis Rhizosphere soil samples were collected from three independent experiments on Chinese cabbage grown for 56 d. We compared the microbial diversity in fertilizer-treated soils using 16s rRNA sequencing analysis following previously described methods [ 15 ]. Microbial DNA was sequenced using a NovaSeq 6000 system (Illumina, San Diego, CA, USA) after constructing a library using the Illumina 16S Metagenomic Sequencing Library [ 31 ]. The raw sequencing data was analyzed using the Cutadapt program [ 32 ] and R packages DADA2 [ 33 ], QIME [ 34 ], dplyr [ 35 ], taxa [ 36 ], ape [ 37 ], ggrepel [ 38 ], pyloseq [ 39 ], DESeq2 [ 40 ], vegan [ 41 ], ggsignif [ 42 ], and ggplot2 [ 43 ]. The Amplicon Sequece Variant (ASV) sequence was identified using the BLAST + program as previously described [ 44 ] and the National Center for Biotechnology Information (NCBI) 16S microbial database. We evaluated significant differences using the R package Agricolae and Duncan’s test and considered significance at P ≤ 0.05. Canonical correspondence was evaluated using the R package CCA ( https://cran.r-project.org/web/packages/CCA/index.html ). 3 Results Chemical components The chemical components of the CLF, FLM, and LBF used in the experiments were based on an EC of 1.5 (Table 1 ). In both liquid fertilizers, we confirmed a constant EC value (1.5), indicating that they were suitable treatments. Among the different liquid fertilizers, a significantly ( P ≤ 0.05) higher pH was observed in CLF (7.53) and FLM (7.63) compared to LBF (7.07). In the liquid fertilizers, the FLM (75.6 mg/kg) and LBF (80.42 mg/kg) had relatively high amounts of nitrate nitrogen (NO 3 − -N). The highest amounts of exchangeable cations were observed in the CLF for K (242 mg/kg) and in the FLM for Na (114 mg/kg) and Ca (109 mg/kg). The soil pH of the soil was significantly increased in the FLM (7.17) and LBF (7.03) groups compared to the CLF group (6.76). The NH 4 + -N content in the soil was highest in the CLF group (200.81 mg/kg) and lowest in the LBF group (114.79 mg/kg). The FLM group (184.49 mg/kg) and LBF group (182.34 mg/kg) had relatively high amounts of nitrate nitrogen (NO 3 − -N) in the soil as well as in the liquid fertilizers. The highest amounts of exchangeable cations were observed in the soils of the CLF group for K (0.15 mg/kg) and Ca (109 mg/kg) and in the FLM group for Na (0.53 mg/kg). However, EC, total N, total P, and OM did not differ significantly among the liquid fertilizers tested. We observed changes in pH and NH 4 + -N according to the quantitative order of the chemical components between the liquid fertilizers and soils after liquid fertilizer treatments. Overall, we have identified the lower pH and highest amounts of NH 4 + -N, K, and Ca in the soil of CLF group. While, the FLM and LBF groups had higher amounts of NO 3 − -N and Na compared to the CLF group. Table 1 Chemical components of different liquid fertilizers (chemical liquid fertilizer [CLF], fermented liquid manure [FLM], and liquid Bio-fertilizer [LBF]). with electrical conductivity of 1.5 and the chemical components of soil after treatment. Fertilizers Soil after treatments CLF FLM LBF CLF FLM LBF pH 7.53 (± 0.06 ) a 6.63 (± 0.04 ) a 7.07 (± 0.08 ) b 6.76 (± 0.09 ) b 7.17 (± 0.05 ) a 7.03 (± 0.03) a EC (dS/m) 1.57 (± 0.08 ) a 1.59 (± 0.11 ) a 1.53 (± 0.09 ) a 0.12 (± 0.02 ) a 0.11 (± 0.01 ) a 0.10 (± 0.01 ) a NH 4 + -N (mg/kg) 578 (± 25.05) a 590 (± 17.67 ) a 560 (± 25.13 ) a 1279.61 (± 142.77 ) a 1266.67 (± 153.93 ) a 1158.01 (± 20.17 ) a NO 3 − N (mg/kg) 64.96 (± 7.21 ) a 46.2 (± 9.25 ) a 74.29 (± 5.53 ) a 200.81 (± 14.58 ) a 140.10 (± 18.53 ) ab 114.79 (± 14.21 ) b P (mg/kg) 14.66 (± 5.04 ) a 19.61 (± 9.17 ) a 37.26 (± 7.1 ) a 1115.74 (± 71.23 ) a 1028.84 (± 65.46 ) a 1110.33 (± 36.53 ) a K (mg/kg) 242 (± 2.57 ) a 212 (± 3.33 ) b 177 (± 3.88 ) c 0.15 (± 0.006 ) a 0.13 (± 0.007 ) b 0.14 (± 0.003) b Na (mg/kg) 47.5 (± 1.23 ) c 114 (± 1.8 ) b 78.53 (± 0.75 ) a 0.22 (± 0.02 ) b 0.53 (± 0.05 ) a 0.51 (± 0.04 ) a Ca (mg/kg) 37.47 (± 0.34 ) c 109 (± 1.04 ) a 90.79 (± 1.27 ) b 9.62 (± 0.4 ) a 8.08 (± 0.4 ) b 8.50 (± 0.27 ) ab OM (%) 0.02 (± 0.03 ) b 0.08 (± 0.06 ) a 0.11 (± 0.04 ) a 1.69 (± 0.34 ) a 1.45 (± 0.24 ) a 1.63 (± 0.19 ) a Lowercase letters represent significant differences ( P < 0.05) between groups as determined by Duncan’s test. Effect of different liquid fertilizers on plant growth We observed the diverse effects of CLF, FLM, and LBF on plant development (Fig. 1 ). The phenotypes of Chinese cabbage grown with different liquid fertilizers were distinctly different; in particular, CLF and LBF led to improved leaf growth and plant width compared with FLM (Fig. 1 A). We observed a significant difference in the length of plants grown for 29 d in the FLM group, with the FLM group showing significantly lower growth compared to the other groups. Chinese cabbage had the largest increase in length on day 57 in the CLF group (24.88 cm), followed by the LBF (22.55 cm) and FLM (21.93 cm) groups (Fig. 1 B). Through the entire growth period, the width and length of leaves increased the most in the CLF group (14.35 cm in width and 24 cm in length on day 57), followed by the LBF group (13.22 cm in width and 22.1 cm in length on day 57) and the FLM group (11.66 cm in width and 20.85 cm in length on day 57). Chlorophyll concentrations significantly increased in the CLF group (41.48) from 49 to 57 days, followed by the LBF (37.2) and FLM groups (32.08). The fresh and dry weights of plant tissues, except roots, in the CLF group (362.33 g fresh weight, 16.95 g dry weight) were heavier than those of the LBF (294.22 g fresh weight, 13.43 g dry weight) and FLM (248.77 g fresh weight, 10.86 g dry weight) groups. LBF treated plants showed improved growth responses while the other fertilizers had more moderate effects. We measured the color of Chinese cabbage leaves after harvest using the Hunter L, a, b color scale (Fig. 2 ). Hunter L* values, in which a low number indicates reduced brightness, were lower in the CLF group (40.57 L*) than in the LBF (42.13 L*) and FLM (43.77 L*) groups. Thus, Chinese cabbage leaves were darker in color in the CLF group and lighter in the LBF and FLM groups. Hunter a* values, which indicate the chromatic value from positive (red) to negative (green), were − 13.28 a* in CLF, -14.86 a* in FLM, and − 14.05 a* in LBF, indicating a relatively low leaf content in the FLM group. The Hunter b* value indicates yellowness (positive number) or blueness (negative number). The highest b* value was observed in the FLM group (20.06 b*) and the lowest was observed in the CLF group (16.37 b*), indicating that the FLM group had a higher yellow color content. Antioxidant activities To survey the biomass changes induced by the liquid fertilizers, we analyzed the sugar and ascorbic acid contents and antioxidant activities in the leaves of the CLF, FLM, and LBF treated plants (Fig. 3 ). The LBF group had a significantly higher sugar content (6.33 mg/mL) compared to the other groups (CLF group: 5.2 mg/mL; FLM group: 5.47 mg/mL) (Fig. 3 A). To evaluate vitamin C content, we measured ascorbic acid, a water-soluble vitamin, in the leaves and stems of Chinese cabbage. Across all plants, the ascorbic acid content was higher in the leaves than in the stems and it was significantly higher in the leaves of the FLM and LBF groups compared to the CLF group and significantly higher in the stems of the CLF and FLM groups compared to the LBF group. To survey the different effects of fertilizers on antioxidant activities, we compared the antioxidant content of Chinese cabbage leaves (Fig. 3 B). We found relatively high levels of antioxidants in the LBF group including total polyphenol content (2.89 mg GAE/mL for CLF, 4.56 mg GAE/mL for FLM, and 5.77 mg GAE/mL for LBF), flavonoid content (17.11 mg QE/mL for CLF, 19.43 mg QE/mL for FLM, and 22.55 mg QE/mL for LBF), DPPH radical scavenging activity (35.7% for CLF, 54.74% for FLM, and 63.96% for LBF), nitrite scavenging activity (65.36% for CLF, 63.31% for FLM, and 73.87% for LBF), reducing power (OD of 1.08 for CLF, 1.7 for FLM, and 2.21 for LBF), and ferric reducing antioxidant power (FRAP; 15.3 µMFe2+/mL for CLF, 23.61 µMFe2+/mL for FLM, and 28.61 µMFe2+/mL for LBF). However, nitrite scavenging activity was not significantly different between the fertilizer groups. The CLF group showed significantly weaker antioxidant activities than the other groups, except for the nitrite scavenging activity. These results indicate the possible utilization of LBF as a liquid bio-fertilizer for the improvement of plant biomass in agriculture. Soil microbial composition To survey the effects of different liquid fertilizers on soil microbiomes, we performed 16S rRNA sequencing analysis of the bacteria in each group (Fig. 4 ). The number of ASVs was lower in the untreated soil (77) as control than in the others (147.83 in CLF, 146 in FLM, and 157 in LBF; Fig. 4 A). The LBF group showed relatively high alpha diversity in both soils; however, there was no significant differences among the liquid fertilizer-treated soils. The ASVs of the soil microbiomes clustered into 27 phyla of bacteria (Fig. 4 B). Ten of the phyla ( Acidobacteria, Actinobacteria, Bacteroidetes , Chloroflexi , Firmicutes , Gemmatimonadetes , Nitrospirae , Planctomycetes , Proteobacteria , and Verrucomicrobia ) comprised over 1,000 of the ASVs detected from the soil bacteria in each sample; the ASVs of Actinobacteria and Proteobacteria comprised a larger percentage of soil bacterial DNA (Fig. 4 B and Fig. S3 ). A relatively small number of ASVs were found from Acidobacteria and Planctomycetes in the CLF group and from Bacteroidetes , Gemmatimonadetes , and Nitrospirae in the non-treated group (Fig. S3 ). Phyla accounting for a large amount of bacterial DNA were Firmicutes in the untreated group and Verrucomicrobia in the FLM and LBF groups. To demonstrate the microbial changes induced by liquid fertilizers, we compared the significant differences (FDR < 0.05) in ASVs of soil bacteria at the genus level (Fig. 4 C). Seventeen genera from seven bacterial phyla were increased in the CLF group compared to the control group; in particular, many CLF-induced genera were included in the phyla Actinobacteria (nine genera) and Proteobacteria (eight genera). Arthrobacter and Streptomyces of the phylum Actinobacteria , Neobacilus of the phylum Firmicutes , and Litorilinea of the phylum Chloroflexi were decreased by CLF compared to FLM and LBF. Twenty-three genus (14 genera increased by FLM and nine genera decreased by FLM) from six phyla were differentially distributed in the FLM group compared with the control group. Two genera ( Luteolibacter in the phylum Verrucomicrobia and Actinoplanes in the phylum Actinobacteria ) showed significant differences in bacterial composition between FLM and control groups. FLM increased the abundance of many genera of the phylum Proteobacteria , while Priestia and Litchfieldia of the phyla Firmicutes , Nocardia, Nocardioides, Actinophytocola, Conexibacter, Pseudarthrobacter , and Aeromicrobium of the phylum Actinobacteria , and Sphingomonas of the phylum Proteobacteria were decreased in the FLM group. When comparing the LBF and control groups, Luteolibacter (phylum Verrucomicrobia ) and Actinoplanes (phylum Actinobacteria ) showed relatively large changes in bacterial composition, along with differences in distribution between FLM and control groups. Many bacterial genera that were increased by LBF from the phyla Actinobacteria (4 genera) and Proteobacteria (5 genera). Hydrogenispora of phylum Firmicutes and Trichocoleus of the phylum Cyanobacteria were decreased by LBF compared to control. Bacteria significantly correlated with chemical compositions NO 3 − -N, K, Na, and Ca from fertilizers, pH, and NO 3 − -N and exchangeable Na from soil were significantly correlated with the community of differentially distributed bacteria in the soils (Fig. 5 A). We hypothesized that the seven chemical compositions of the fertilizers or soils influenced soil bacterial changes. Thus, we conducted a canonical correspondence analysis (CCA) between the seven chemical compositions and the differentially distributed bacteria (Fig. 5 B) and evaluated the significance of the correlation (Table 2 ). Finally, we identified the significantly correlated genera among the five phyla. We found that six genera ( Terracoccus , Virgibacillus , Lysobacter , Mesorhizobium , Nitrospira , and Rhizobium ) were significantly correlated with the K content of the fertilizer. Interestingly, the six K-related genera had significant positive or negative correlations with one of the six chemical compositions, except for K. The CCA plot was divided into four quadrants (I-IV). The FLM group was located close to Na (soil) in quadrant I, while the LBF group was located close to NO 3 − -N (fertilizer), Na (fertilizer), Ca (fertilizer), pH (soil), and NO 3 − -N (soil) in quadrant II (Fig. 5 B). K (fertilizer) was correlated with the CLF group in quadrant IV. This suggests that the chemical composition of the fertilizers led to distinct bacterial communities. Table 2 Soil bacteria significantly correlated with chemical compositions determined by the Mantel test. Phylum Genus Na_F K_F NO3_F Ca_F pH_S NO3_S Na_S Actinobacteria Actinoplanes 0.77* -0.21 0.51 0.65 0.63 0.57 0.49 Nocardioides -0.77* 0.45 -0.7* -0.77* -0.95*** -0.61 -0.72* Pedococcus -0.7* 0.66 -0.84** -0.81** -0.94*** -0.7* -0.78* Pseudarthrobacter -0.45 0.56 -0.59 -0.53 -0.81** -0.53 -0.62 Terrabacter -0.58 0.67 -0.71* -0.7* -0.76* -0.65 -0.78* Terracoccus -0.85** 0.75* -0.9** -0.93*** -0.81** -0.89** -0.93*** Chloroflexi Litorilinea 0.86** -0.5 0.75* 0.88** 0.66 0.8** 0.94*** Firmicutes Neobacillus 0.66 -0.9*** 0.91*** 0.82** 0.7* 0.92*** 0.88** Virgibacillus 0.27 -0.81** 0.64 0.45 0.48 0.56 0.3 Proteobacteria Lysobacter -0.78* 0.83** -0.94*** -0.92*** -0.73* -0.94*** -0.97*** Massilia 0.81** -0.24 0.59 0.78* 0.59 0.52 0.64 Mesorhizobium -0.22 0.74* -0.69* -0.49 -0.58 -0.47 -0.55 Nitrosospira -0.72* 0.74* -0.9** -0.85** -0.89** -0.79* -0.87** Rhizobium -0.72* 0.81** -0.89** -0.86** -0.57 -0.92*** -0.9*** Rhizorhabdus 0.79* -0.5 0.63 0.78* 0.83** 0.66 0.71* Sphingomonas -0.74* -0.23 -0.26 -0.51 -0.48 -0.36 -0.4 Sphingopyxis -0.47 0.65 -0.72* -0.6 -0.79* -0.58 -0.69* Verrucomicrobia Luteolibacter 0.76* -0.43 0.78* 0.74* 0.75* 0.75* 0.59 Fold change value was calculated between treatments and control (log 2 [treatments / control]) by DESeq2 (NS, non-significance). The significance of Pearson’s correlation coefficient (PCC) was determined by n-2 degrees of freedom (* P < 0.05, ** P < 0.01, *** P < 0.001) To determine the specific distribution of bacteria with each fertilizer treatment, we compared the relative ASV abundance compared to the control (Fig. 5 C). Litorilinea was decreased in the CLF group, but not in the FLM and LBF groups. The FLM group showed a specific decrease in Sphingomonas and Nocardioides . LBF showed increases in Sphingomonas and Nocardioides similar to CLF, and an increase in Litorilinea similar to FLM, suggesting that the bacterial communities were in the middle of CLF and FLM. 4 Discussion NO 3 − -N and Na from the fertilizer and soil correlated with the FLM and LBF groups in this study. The ratio of nitrogen sources (NH 4 +-N or NO 3 − -N) in chemical fertilizers led to distinct plant growth. Low ammonium (25%) and high nitrate (75%) have been shown to increase the plant growth of flowering Chinese cabbage (cv. Lvbao70, cv. Youlv80, and cv. Chixin No.2), while high ammonium (75%) and low nitrate (25%) had no significant effect on improving plant growth [ 45 ]. Furthermore, nitrate, in comparison with ammonium, has been shown to significantly increase the water content and leaf area of spinach, sunflower, and pea plants [ 46 ]. Although FLM and LBF had higher nitrate-nitrogen contents, the CLF group showed a stronger promotion of Chinese cabbage plant growth. We found higher amounts of Na in FLM and LBF liquid fertilizers and treated soils. In particular, the exchangeable Na in the liquid fertilizer was higher in FLM than in LBF. High levels of exchangeable Na in the soil have been shown to have negative effects on plant growth and soil nutrition [ 47 ]. For example, exchangeable Na leads to reduced alfalfa and cotton growth according to its percentage in the soil [ 48 ]. The amount of exchangeable Na was shown to increase in the soil during a second cropping season using liquid fertilization developed from pig slurry compared to using chemical fertilizer [ 49 ]. Thus, we suggest that the high amount of exchangeable Na + in FLM and LBF inhibited plant development in Chinese cabbage. The organic fertilizers FLM and LBF led to increased levels of sugars, ascorbic acid, and antioxidants within the plants. Liquid organic fertilizer was found to increase the ascorbic acid and crude protein content in bell peppers (cv. Red Wonder F1) compared to mineral fertilizer [ 50 ]. Furthermore, the total content of phenolics, flavonoids, and glucosinolates in broccoli were increased by the additional application of organic fertilizers [ 51 ]. Chlorella extracts have been shown to promote plant growth in pepper ( Capsicum annuum ) plants [ 52 ]. For example, plant height, leaf area, and fruit and shoot weights of pepper plants were increased after supplying Chlorella extracts for 21 days compared to the control. Furthermore, the antioxidant enzymes superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were increased with Chlorella extract treatment in pepper plants, suggesting the ability of Chlorella extracts to promote plant growth. On an organic farm, Chlorella culture treatments led to improved height, leaf width, fresh weight, and yield of Chinese chives and improved leaf thickness, leaf number, fresh weight, yield, and mineral content of spinach [ 53 ]. Chlorella has economic value because of its diverse advantages such as plant growth promotion and functional food production in the application of plant bio-stimulants, including plant growth-promoting bacteria, microalgae, and mycorrhiza [ 54 , 55 ]. Chlorella minutissima extracts had significantly higher DPPH RSA, FRAP, and iron chelating activity (ICA) values than those of other microalgal species ( Dunaliella salina, Isochrysis galbana, Nannochloropsis oculate , and Tisochrysis lutea ) [ 56 ]. However, the effects of a Chlorella culture solution as a liquid bio-fertilizer on plant antioxidant activity have not yet been fully elucidated. We hypothesized that Chlorella culture liquid fertilizer could increase antioxidant levels in Chinese cabbage. The LBF group showed greater improvements in antioxidant levels compared to the CLF and FLM groups. We found that Litorilinea was significantly decreased in the CLF group and increased in the FLM and LBF groups. Litorilinea is an aerobic gram-negative bacterium [ 57 ]. Thus, it is possible that Litorilinea increased due to anaerobic fermentation during the composting of the livestock manure used as a liquid fertilizer. Nocardioides is a genus of gram-positive aerobic bacteria belonging to the family Nocardioidaceae, whereas Sphingomonas is a genus of gram-negative aerobic bacteria belonging to the family Sphingomonadaceae [ 58 , 59 ]. Anaerobic fermentation for FLM production led to a decrease in Nocardioides and Sphingomonas . These bacteria contribute to nutrient cycling during the decomposition of complex biomasses and play a role in nitrogen fixation, which benefits plant growth and production [ 60 , 61 ]. Sphingomonas can promote plant growth under normal and/or drought stress conditions owing to its contribution to root developmental plasticity [ 62 , 63 , 64 ]. In the soil, a lack of Nocardioides and Sphingomonas may have led to the decreased growth rate of the FLM treated plants compared to the CLF and LBF groups. The newly developed LBF was generated by culturing Chlorella in an organic liquid fertilizer using livestock manure without a chemical medium. Recycling organic liquid fertilizers instead of chemical cultures is a cost-effective solution for the production of Chlorella biostimulants. 5 Conclusion Here, we observed the effects of CLF and organic liquid fertilizers on plant growth, antioxidants, and soil microorganisms. To survey the changes of chemical components by different fertilizers, the amounts of NO 3 − -N and Na were significantly different between the CLF and organic liquid fertilizers (FLM and LBF) groups. This result indicated that the plant growth of Chinese cabbage was inhibited in the FLM and LBF groups due to Na increase and the unsuitable ratio of NH 4 +-N and NO 3 − -N. However, LBF has improved the growth and substances of plants compared to FLM as an organic medium for Chlorella cultivation, suggesting the utilization of livestock manure. Finally, we have identified the distinct soil bacterial communities among different fertilizer-treated groups. For example, Litorilinea as anaerobic bacteria was decreased in the CLF group compared to FLM and LBF groups, which have needed anaerobic fermentation to produce livestock manure as raw material. Furthermore, Chinese cabbage may reduce plant growth in the FLM group by decreasing plant growth promoting bacterial species such as Nocardioides and Sphingomonas. Overall, the CLF affected to the highest growth rate of Chinese cabbage, while, the accumulation of plant substances was increased by organic liquid fertilizers (FLM and LBF). We identified that the Chlorella cultivation for LBF led to increases in plant growth and antioxidants. Thus, the development of organic bio-liquid fertilizers such as LBF will contribute to the construction of natural recycling systems through the application of organic fertilizers from livestock manure on organic farms. Abbreviations CLF, chemical liquid fertilizer; FLM, fermented liquid Manure; LBF, liquid bio-fertilizer; PGPB, plant growth-promoting bacteria; EC, electrical conductivity; OM, organic matter; ASV, Amplicon Sequence Variant; NCBI, National Center for Biotechnology Information; FRAP, ferric reducing antioxidant power; CCA, canonical correspondence analysis; SOD, superoxide dismutase; POD, peroxidase; CAT, catalase; ICA, iron chelating activity; Declarations Competing interests The authors declare no competing interests. Consent for publication Not applicable. Ethics approval and consent to participate Our research did not involve any studies with human or animal subjects. Experimental research on the plants complies with relevant institutional, national, and international guidelines and legislation and used for research and development. Funding This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry(IPET) and Korea Smart Farm R&D Foundation(KosFarm) through Smart Farm Innovation Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs(MAFRA) and Ministry of Science and ICT(MSIT), Rural Development Administration(RDA) (421046-03) Availability of data and materials The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: BioProject, PRJNA982574 Author Contributions J.K.L. and S.G.H. conceived of and designed the study. J.K.L., N.Y.J., T.Y.L.L., W.Y.J., K.W.K., H.S.C., B.OL., S.R.K., and M.G.L. performed the field sampling. J.K.L., N.Y.J., S.Y.S., S.R.K., M.G.L., and S.G.H. collected and analyzed the data. J.K.L. and S.G.H. wrote the manuscript. J.K.L. and N.Y.J. provided reagents and materials. All authors contributed critically to the article, drafts, and revisions and gave final approval for publication. Acknowledgements Not applicable. Author details 1 College of Life and Environment Science, Sangji University, Wonju-si 26339, Republic of Korea. 2 Department of Smart-Farm, Sangji University, Wonju-si 26339, Republic of Korea. 3 Hoengseong Agricultural technology extension center, Hoengseong 25208, Republic of Korea. 4 Hanbio Incoporated. Hoengseong 25249, Republic of Korea. 5 Industry Academic Cooperation Foundation Resource Recycle Engineering Lab, Sangji University, Wonju-si 26339, Republic of Korea. 6 Department of Smart Life Science, Sangji University, Wonju-si 26339, Republic of Korea References Maltas A, Charles R, Jeangros B, Sinaj S. Effect of organic fertilizers and reduced-tillage on soil properties, crop nitrogen response and crop yield: Results of a 12-year experiment in Changins, Switzerland. Soil Tillage Res. 2013;126:11–8. Tilman D. 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(A) Phenotypes by different fertilizer treatments (chemical liquid fertilizer (CLF), fermented liquid manure (FLM), and liquid Bio-fertilizer (LBF)). (B) The plant length, chlorophyll content, leaf length and width, and fresh and dry weight in the plants at 57 days after planting. The line colors represent the different fertilizer treatments (red; CLF, black; FLM, blue; LBF). The values are the mean ± standard deviation (n = 9). Lowercase letters indicate significant differences (\u003cem\u003eP\u003c/em\u003e \u0026lt;0.05) between groups by the Duncan test.\u003c/p\u003e","description":"","filename":"OnlineFigure1..png","url":"https://assets-eu.researchsquare.com/files/rs-2964376/v1/7876fc6d300a4a991fdd3262.png"},{"id":38666704,"identity":"48c1ffae-d3f7-4cab-9d82-edf48cabb42a","added_by":"auto","created_at":"2023-06-16 14:45:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":87744,"visible":true,"origin":"","legend":"\u003cp\u003eColor analysis in the leaves of Chinese cabbage after harvest using the Hunter (L, a, b) system. Chemical liquid fertilizer, CLF; fermented liquid manure, FLM; and liquid Bio-fertilizer, LBF. The values are the mean ± standard deviation (n = 9). Lowercase letters indicate significant differences (\u003cem\u003eP\u003c/em\u003e \u0026lt;0.05) between groups by the Duncan test.\u003c/p\u003e","description":"","filename":"OnlineFigure2..png","url":"https://assets-eu.researchsquare.com/files/rs-2964376/v1/33f88e73ba610b24c9bc0194.png"},{"id":38670469,"identity":"4cef921f-a718-48d6-a232-afc6d2e6747f","added_by":"auto","created_at":"2023-06-16 15:01:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":146250,"visible":true,"origin":"","legend":"\u003cp\u003eDifferences of functional plant materials with different fertilizer supplies. (A) The contents of sugar and ascorbic acid. (B) The contents and activities of antioxidants. The analysis used Chinese cabbage leaves. Chemical liquid fertilizer, CLF; fermented liquid manure, FLM; and liquid Bio-fertilizer, LBF. The values are the mean ± standard deviation (n = 3). Lowercase letters indicate significant differences (\u003cem\u003eP\u003c/em\u003e \u0026lt;0.05) between groups by the Duncan test.\u003c/p\u003e","description":"","filename":"OnlineFigure3..png","url":"https://assets-eu.researchsquare.com/files/rs-2964376/v1/aa3adaffd711e7a7732cef23.png"},{"id":38668739,"identity":"5a531e8f-ddb3-468c-81be-5cb3d76deab4","added_by":"auto","created_at":"2023-06-16 14:53:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":172266,"visible":true,"origin":"","legend":"\u003cp\u003eAmplicon sequence variant (ASV) analysis of the bacteria in the soils treated with different fertilizers. (A) Alpha diversity of the bacterial population in each sample using the InvSimposn method. Lowercase letters represent the significant differences (\u003cem\u003eP\u003c/em\u003e \u0026lt;0.05) between groups by Tukey’s HSD test. (B) Bacterial communities of each sample at the phylum level. Box colors represent the phylum of bacteria. (C) The quantity changes of bacterial DNA in each sample compared to the control. The dot colors indicate the phylum of the bacterial species. Chemical liquid fertilizer, CLF; fermented liquid manure, FLM; and liquid Bio-fertilizer, LBF.\u003c/p\u003e","description":"","filename":"OnlineFigure4..png","url":"https://assets-eu.researchsquare.com/files/rs-2964376/v1/59f70cc522f0a4bd7631aa12.png"},{"id":38666707,"identity":"3d8fa886-ac9d-4325-9e46-083a8267d6a2","added_by":"auto","created_at":"2023-06-16 14:45:35","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":293464,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation of soil properties with bacterial community composition. (A) Mantel test between soil properties on bacterial community composition. Significant results are indicated by *\u003cem\u003eP \u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt;0.01, NS = no significance. (B) Canonical correspondence analysis (CCA) relating soil bacteria genus group. (C) The relative abundances of significantly correlated bacteria in CLF, FLM, and LBF treated soils compared to control. Circle size represents the number of bacteria for ASV. The color indicates the log 2-fold change of ASV with p-adjust \u0026lt;0.05. Arrows represent the specific bacteria in each treatment. Chemical liquid fertilizer, CLF; fermented liquid manure, FLM; and liquid Bio-fertilizer, LBF.\u003c/p\u003e","description":"","filename":"OnlineFigure5..png","url":"https://assets-eu.researchsquare.com/files/rs-2964376/v1/90dc56e9502619ff8045a583.png"},{"id":79623132,"identity":"3ff7f2ae-066b-47e9-9c25-4221deaed12f","added_by":"auto","created_at":"2025-04-01 00:01:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1852782,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2964376/v1/f74f8257-18ab-4552-b5a8-8433df1ef0ac.pdf"},{"id":38666709,"identity":"de070230-daf2-4f58-b1ce-65c41296c358","added_by":"auto","created_at":"2023-06-16 14:45:35","extension":"tif","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":6642792,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure1..tif","url":"https://assets-eu.researchsquare.com/files/rs-2964376/v1/6c5eedfbe8a6b41a274da337.tif"},{"id":38666708,"identity":"af7ac329-66f8-4063-9409-67ebaefa7a3f","added_by":"auto","created_at":"2023-06-16 14:45:35","extension":"tif","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":837258,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure2..tif","url":"https://assets-eu.researchsquare.com/files/rs-2964376/v1/fe008f15414831d18495a147.tif"},{"id":38666710,"identity":"8b9ece14-015d-4195-aaad-272d2650cff2","added_by":"auto","created_at":"2023-06-16 14:45:35","extension":"tif","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":3097178,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure3..tif","url":"https://assets-eu.researchsquare.com/files/rs-2964376/v1/c0980c4fa62d536addfb4961.tif"},{"id":38666705,"identity":"12d6f081-66fd-4544-a7ec-928ed9352ab4","added_by":"auto","created_at":"2023-06-16 14:45:35","extension":"docx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":15887,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-2964376/v1/52073f69f501ea6a0e67326a.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Influence of organic liquid fertilizer developed from livestock manure on the growth, antioxidant activities, and soil microbial populations of Chinese cabbage","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eChemical fertilizers have played an important role in maintaining sufficient crop production owing to the increased food demand of a growing human population, despite decreasing farmland area [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, the excessive use of chemical fertilizers can cause environmental pollution. For example, 50% of the nitrogen and 90% phosphorus of applied fertilizer in the cultivation region are released into the atmosphere or water sources. These released materials lead to the formation of greenhouse gases in the atmosphere, saline contamination in soil, and the eutrophication in the aquatic ecosystems [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Diverse organisms, such as soil microbes, humans, plants, and animals, in natural ecosystems have been adversely affected by the imbalance of soil nutrient ratios due to the use of chemical fertilizers [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Therefore, environmentally friendly organic fertilizers have been suggested for sustainable agriculture [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The agricultural traits of kiwi plants (\u003cem\u003eActinidia deliciosa\u003c/em\u003e) were improved, with an increase in crop production and plant nutrition, using organic fertilizers with crop residues and other biological wastes [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Furthermore, the availability of organic manure improved the growth, yield, and mineral content of okra (\u003cem\u003eAbelmoschus esculentus\u003c/em\u003e) in comparison with NPK 15-15-15 fertilizer [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In a previous study, we found that the growth of maize was enhanced by supplying a mix of Hanwoo (Korean cattle) manure and chemical fertilizer compared to chemical fertilizer alone [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Studies on organic fertilizers have provided useful information for the development of sustainable agriculture [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMicrobiome soil inoculation, which can replace synthetic farm inputs, has positive effects on the soil environment and plant growth; many studies on soil microorganisms have been conducted in recent years [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Plant growth-promoting bacteria (PGPB) have direct and/or indirect effects on the solubilization of soil nutrients (P, K, and Fe) and enhance tolerance to pests and diseases [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. For example, one type of PGPB, rhizobacteria, improves water and nutrient uptake and tolerance to abiotic and biotic stress in the interaction between the microbiome and the plant [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Bio-fertilizers, including \u003cem\u003eBacillus megaterium\u003c/em\u003e var. \u003cem\u003ephosphaticum\u003c/em\u003e and \u003cem\u003eAzotobacter chroococcum\u003c/em\u003e, increased the dry weight of the roots and shoots of maize compared to untreated plants [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Some studies have attempted to understand the combined effects of two or more microbes on crop cultivation; however, the exact mechanisms underlying the diverse biological effects of soil microorganisms remain unclear [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. \u003cem\u003eChlorella\u003c/em\u003e is a photosynthetic microorganism with a high potential for the production of useful metabolites [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The hormones auxin and cytokinin, which regulate plant growth and development, are increased during the cultivation of \u003cem\u003eChlorella\u003c/em\u003e and interact with plant metabolism [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. These results suggest that the use of bio-liquid fertilizers with microorganisms will be useful for the improvement of crop production and soil amendment. Here, we attempted to produce the organic bio-liquid fertilizer by \u003cem\u003eChlorella\u003c/em\u003e cultivation with media developed from livestock manure.\u003c/p\u003e \u003cp\u003eThe Chinese napa cabbage (\u003cem\u003eBrassica rapa\u003c/em\u003e subsp. \u003cem\u003epekinensis\u003c/em\u003e (Lour.) Hanelt) is the most popular vegetable in East Asia, China, and Korea and is used for making kimchi, a traditional Korean fermented food. Chinese cabbage is a highly adaptable plant that is grown extensively worldwide and contains many nutrients, including vitamin C, dietary flavonols, crude fiber, and carotenoids [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. This study aimed to enhance the quantity and quality of greenhouse crops, through the application of liquid fertilizers. We observed the effects of different organic liquid fertilizers on plant growth, antibiotic activities of Chinese cabbage, and soil bacterial communities compared to chemical fertilizers. This study provides useful information for realizing environment-friendly agriculture in greenhouses.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cp\u003ePlant growth and fertilizers\u003c/p\u003e \u003cp\u003eChinese napa cabbage (\u003cem\u003eBrassica rapa\u003c/em\u003e subsp. \u003cem\u003epekinensis\u003c/em\u003e (Lour.) Rupr.) seeds were obtained from the Danong Company (Gyeonggi-do, Korea). The seeds were grown in soil-filled pots (18 \u0026times; 18 \u0026times; 30 cm) in the greenhouse of the Agricultural Technology center (located in Hoengseong County, Gangwon-do, Republic of Korea) under an average temperature of 25℃ and an average humidity of 65%. Plants treatments were performed with three independent replicates of each experimental unit. NPK fertilizer (21-17-17) used as the chemical liquid fertilizer treatment (CLF) was obtained from the Namhae Chemical Corporation (Jeollanam-do, Korea). The fermented liquid manure (FLM) was created from livestock manure, which was provide from hog farm (37\u0026deg;30'33.1948211615924\"N 128\u0026deg;04'56.5629151080066\"E) after the livestock excretion had decomposed for one year. The organic medium developed from livestock manure was produced following a previously described method [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] and was used to grow \u003cem\u003eChlorella fusca\u003c/em\u003e, which was provided by the National Institute of Agricultural Sciences, Republic of Korea. \u003cem\u003eChlorella\u003c/em\u003e was grown in an incubator (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) to a concentration of 10\u003csup\u003e7\u003c/sup\u003e cells/mL at a mean temperature of 28℃. The incubator was comprised of LED modules (FNB-240LED; F\u0026amp;B Nature, Chungju, Korea) with red and blue light (16 h/8 h day/night cycle) and an air supply providing 0.1 m\u003csup\u003e3\u003c/sup\u003e air/m\u003csup\u003e3\u003c/sup\u003e\u0026middot;min [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The \u003cem\u003eChlorella\u003c/em\u003e-incubated medium was filtered using a tubular continuous centrifuge (J-1050A; Hanil Sci-Med, Chungcheongbuk-do, Korea) at 12, 000 \u0026times; g to obtain the liquid bio-fertilizer (LBF). The liquid fertilizers were treated with 1.5 mS/cm electrical conductivity (EC). After sowing the seeds, liquid fertilizers were supplied twice per day (50 mL at both 9:00 a.m. and 6:00 p.m.) using a dropper.\u003c/p\u003e \u003cp\u003eSurvey of plant growth, chemical components, and antioxidants\u003c/p\u003e \u003cp\u003ePlant growth (plant length, fresh and dry weight, leaf color and length, and chlorophyll content), chemical components of the soil and liquid fertilizer (pH, EC, total N, NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N, NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N, P, K, Na, Ca, and organic matter [OM]), and antioxidants (total polyphenol and flavonoid contents, 2,2-Diphenyl-1-picrylhydrazyl [DPPH] and nitrite scavenging activity, reducing power, and Ferric Reducing Antioxidant Power [FRAP]) were all analyzed using previously described methods [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], and the plant substances were extracted by methanol. The standard substances were quercetin for total flavonoid and gallic acid for total phenol, DPPH, and FRAP assay, and then the proper standard curves were estimated (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Leaves of Chinese cabbage grown for 56 days were used for antioxidant analysis and color measurement. Colors (L (lightness), a (redness), and b (yellowness)) were measured using a Hunter Lab Colorimeter (ND-300A; Nippon Denshoku, Tokyo, Japan).\u003c/p\u003e \u003cp\u003eSoil microbiome analysis\u003c/p\u003e \u003cp\u003eRhizosphere soil samples were collected from three independent experiments on Chinese cabbage grown for 56 d. We compared the microbial diversity in fertilizer-treated soils using 16s rRNA sequencing analysis following previously described methods [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Microbial DNA was sequenced using a NovaSeq 6000 system (Illumina, San Diego, CA, USA) after constructing a library using the Illumina 16S Metagenomic Sequencing Library [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The raw sequencing data was analyzed using the Cutadapt program [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] and R packages DADA2 [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], QIME [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], dplyr [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], taxa [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], ape [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], ggrepel [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], pyloseq [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], DESeq2 [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], vegan [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], ggsignif [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], and ggplot2 [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The Amplicon Sequece Variant (ASV) sequence was identified using the BLAST\u0026thinsp;+\u0026thinsp;program as previously described [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] and the National Center for Biotechnology Information (NCBI) 16S microbial database. We evaluated significant differences using the R package Agricolae and Duncan\u0026rsquo;s test and considered significance at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05. Canonical correspondence was evaluated using the R package CCA (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cran.r-project.org/web/packages/CCA/index.html\u003c/span\u003e\u003cspan address=\"https://cran.r-project.org/web/packages/CCA/index.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e"},{"header":"3 Results","content":"\u003cp\u003eChemical components\u003c/p\u003e \u003cp\u003eThe chemical components of the CLF, FLM, and LBF used in the experiments were based on an EC of 1.5 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In both liquid fertilizers, we confirmed a constant EC value (1.5), indicating that they were suitable treatments. Among the different liquid fertilizers, a significantly (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05) higher pH was observed in CLF (7.53) and FLM (7.63) compared to LBF (7.07). In the liquid fertilizers, the FLM (75.6 mg/kg) and LBF (80.42 mg/kg) had relatively high amounts of nitrate nitrogen (NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N). The highest amounts of exchangeable cations were observed in the CLF for K (242 mg/kg) and in the FLM for Na (114 mg/kg) and Ca (109 mg/kg). The soil pH of the soil was significantly increased in the FLM (7.17) and LBF (7.03) groups compared to the CLF group (6.76). The NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N content in the soil was highest in the CLF group (200.81 mg/kg) and lowest in the LBF group (114.79 mg/kg). The FLM group (184.49 mg/kg) and LBF group (182.34 mg/kg) had relatively high amounts of nitrate nitrogen (NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N) in the soil as well as in the liquid fertilizers. The highest amounts of exchangeable cations were observed in the soils of the CLF group for K (0.15 mg/kg) and Ca (109 mg/kg) and in the FLM group for Na (0.53 mg/kg). However, EC, total N, total P, and OM did not differ significantly among the liquid fertilizers tested. We observed changes in pH and NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N according to the quantitative order of the chemical components between the liquid fertilizers and soils after liquid fertilizer treatments. Overall, we have identified the lower pH and highest amounts of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N, K, and Ca in the soil of CLF group. While, the FLM and LBF groups had higher amounts of NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N and Na compared to the CLF group.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eChemical components of different liquid fertilizers (chemical liquid fertilizer [CLF], fermented liquid manure [FLM], and liquid Bio-fertilizer [LBF]). with electrical conductivity of 1.5 and the chemical components of soil after treatment.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eFertilizers\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003eSoil after treatments\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCLF\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFLM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLBF\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCLF\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eFLM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eLBF\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.53 (\u0026plusmn;\u0026thinsp;0.06 ) a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.63 (\u0026plusmn;\u0026thinsp;0.04 ) a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.07 (\u0026plusmn;\u0026thinsp;0.08 ) b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.76 (\u0026plusmn;\u0026thinsp;0.09 ) b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7.17 (\u0026plusmn;\u0026thinsp;0.05 ) a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7.03 (\u0026plusmn;\u0026thinsp;0.03) a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEC (dS/m)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.57 (\u0026plusmn;\u0026thinsp;0.08 ) a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.59 (\u0026plusmn;\u0026thinsp;0.11 ) a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.53 (\u0026plusmn;\u0026thinsp;0.09 ) a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.12 (\u0026plusmn;\u0026thinsp;0.02 ) a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.11 (\u0026plusmn;\u0026thinsp;0.01 ) a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.10 (\u0026plusmn;\u0026thinsp;0.01 ) a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N (mg/kg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e578 (\u0026plusmn;\u0026thinsp;25.05) a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e590 (\u0026plusmn;\u0026thinsp;17.67 ) a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e560 (\u0026plusmn;\u0026thinsp;25.13 ) a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1279.61 (\u0026plusmn;\u0026thinsp;142.77 ) a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1266.67 (\u0026plusmn;\u0026thinsp;153.93 ) a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1158.01 (\u0026plusmn;\u0026thinsp;20.17 ) a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003eN (mg/kg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e64.96 (\u0026plusmn;\u0026thinsp;7.21 ) a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e46.2 (\u0026plusmn;\u0026thinsp;9.25 ) a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e74.29 (\u0026plusmn;\u0026thinsp;5.53 ) a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e200.81 (\u0026plusmn;\u0026thinsp;14.58 ) a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e140.10 (\u0026plusmn;\u0026thinsp;18.53 ) ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e114.79 (\u0026plusmn;\u0026thinsp;14.21 ) b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP (mg/kg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14.66 (\u0026plusmn;\u0026thinsp;5.04 ) a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19.61 (\u0026plusmn;\u0026thinsp;9.17 ) a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e37.26 (\u0026plusmn;\u0026thinsp;7.1 ) a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1115.74 (\u0026plusmn;\u0026thinsp;71.23 ) a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1028.84 (\u0026plusmn;\u0026thinsp;65.46 ) a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1110.33 (\u0026plusmn;\u0026thinsp;36.53 ) a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eK (mg/kg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e242 (\u0026plusmn;\u0026thinsp;2.57 ) a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e212 (\u0026plusmn;\u0026thinsp;3.33 ) b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e177 (\u0026plusmn;\u0026thinsp;3.88 ) c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.15 (\u0026plusmn;\u0026thinsp;0.006 ) a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.13 (\u0026plusmn;\u0026thinsp;0.007 ) b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.14 (\u0026plusmn;\u0026thinsp;0.003) b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNa (mg/kg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e47.5 (\u0026plusmn;\u0026thinsp;1.23 ) c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e114 (\u0026plusmn;\u0026thinsp;1.8 ) b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e78.53 (\u0026plusmn;\u0026thinsp;0.75 ) a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.22 (\u0026plusmn;\u0026thinsp;0.02 ) b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.53 (\u0026plusmn;\u0026thinsp;0.05 ) a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.51 (\u0026plusmn;\u0026thinsp;0.04 ) a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCa (mg/kg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e37.47 (\u0026plusmn;\u0026thinsp;0.34 ) c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e109 (\u0026plusmn;\u0026thinsp;1.04 ) a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e90.79 (\u0026plusmn;\u0026thinsp;1.27 ) b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.62 (\u0026plusmn;\u0026thinsp;0.4 ) a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.08 (\u0026plusmn;\u0026thinsp;0.4 ) b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8.50 (\u0026plusmn;\u0026thinsp;0.27 ) ab\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOM (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.02 (\u0026plusmn;\u0026thinsp;0.03 ) b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.08 (\u0026plusmn;\u0026thinsp;0.06 ) a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.11 (\u0026plusmn;\u0026thinsp;0.04 ) a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.69 (\u0026plusmn;\u0026thinsp;0.34 ) a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.45 (\u0026plusmn;\u0026thinsp;0.24 ) a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.63 (\u0026plusmn;\u0026thinsp;0.19 ) a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eLowercase letters represent significant differences (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) between groups as determined by Duncan\u0026rsquo;s test.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eEffect of different liquid fertilizers on plant growth\u003c/p\u003e \u003cp\u003eWe observed the diverse effects of CLF, FLM, and LBF on plant development (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The phenotypes of Chinese cabbage grown with different liquid fertilizers were distinctly different; in particular, CLF and LBF led to improved leaf growth and plant width compared with FLM (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). We observed a significant difference in the length of plants grown for 29 d in the FLM group, with the FLM group showing significantly lower growth compared to the other groups. Chinese cabbage had the largest increase in length on day 57 in the CLF group (24.88 cm), followed by the LBF (22.55 cm) and FLM (21.93 cm) groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Through the entire growth period, the width and length of leaves increased the most in the CLF group (14.35 cm in width and 24 cm in length on day 57), followed by the LBF group (13.22 cm in width and 22.1 cm in length on day 57) and the FLM group (11.66 cm in width and 20.85 cm in length on day 57). Chlorophyll concentrations significantly increased in the CLF group (41.48) from 49 to 57 days, followed by the LBF (37.2) and FLM groups (32.08). The fresh and dry weights of plant tissues, except roots, in the CLF group (362.33 g fresh weight, 16.95 g dry weight) were heavier than those of the LBF (294.22 g fresh weight, 13.43 g dry weight) and FLM (248.77 g fresh weight, 10.86 g dry weight) groups. LBF treated plants showed improved growth responses while the other fertilizers had more moderate effects.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe measured the color of Chinese cabbage leaves after harvest using the Hunter L, a, b color scale (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Hunter L* values, in which a low number indicates reduced brightness, were lower in the CLF group (40.57 L*) than in the LBF (42.13 L*) and FLM (43.77 L*) groups. Thus, Chinese cabbage leaves were darker in color in the CLF group and lighter in the LBF and FLM groups. Hunter a* values, which indicate the chromatic value from positive (red) to negative (green), were \u0026minus;\u0026thinsp;13.28 a* in CLF, -14.86 a* in FLM, and \u0026minus;\u0026thinsp;14.05 a* in LBF, indicating a relatively low leaf content in the FLM group. The Hunter b* value indicates yellowness (positive number) or blueness (negative number). The highest b* value was observed in the FLM group (20.06 b*) and the lowest was observed in the CLF group (16.37 b*), indicating that the FLM group had a higher yellow color content.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAntioxidant activities\u003c/p\u003e \u003cp\u003eTo survey the biomass changes induced by the liquid fertilizers, we analyzed the sugar and ascorbic acid contents and antioxidant activities in the leaves of the CLF, FLM, and LBF treated plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The LBF group had a significantly higher sugar content (6.33 mg/mL) compared to the other groups (CLF group: 5.2 mg/mL; FLM group: 5.47 mg/mL) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). To evaluate vitamin C content, we measured ascorbic acid, a water-soluble vitamin, in the leaves and stems of Chinese cabbage. Across all plants, the ascorbic acid content was higher in the leaves than in the stems and it was significantly higher in the leaves of the FLM and LBF groups compared to the CLF group and significantly higher in the stems of the CLF and FLM groups compared to the LBF group. To survey the different effects of fertilizers on antioxidant activities, we compared the antioxidant content of Chinese cabbage leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). We found relatively high levels of antioxidants in the LBF group including total polyphenol content (2.89 mg GAE/mL for CLF, 4.56 mg GAE/mL for FLM, and 5.77 mg GAE/mL for LBF), flavonoid content (17.11 mg QE/mL for CLF, 19.43 mg QE/mL for FLM, and 22.55 mg QE/mL for LBF), DPPH radical scavenging activity (35.7% for CLF, 54.74% for FLM, and 63.96% for LBF), nitrite scavenging activity (65.36% for CLF, 63.31% for FLM, and 73.87% for LBF), reducing power (OD of 1.08 for CLF, 1.7 for FLM, and 2.21 for LBF), and ferric reducing antioxidant power (FRAP; 15.3 \u0026micro;MFe2+/mL for CLF, 23.61 \u0026micro;MFe2+/mL for FLM, and 28.61 \u0026micro;MFe2+/mL for LBF). However, nitrite scavenging activity was not significantly different between the fertilizer groups. The CLF group showed significantly weaker antioxidant activities than the other groups, except for the nitrite scavenging activity. These results indicate the possible utilization of LBF as a liquid bio-fertilizer for the improvement of plant biomass in agriculture.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSoil microbial composition\u003c/p\u003e \u003cp\u003eTo survey the effects of different liquid fertilizers on soil microbiomes, we performed 16S rRNA sequencing analysis of the bacteria in each group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The number of ASVs was lower in the untreated soil (77) as control than in the others (147.83 in CLF, 146 in FLM, and 157 in LBF; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The LBF group showed relatively high alpha diversity in both soils; however, there was no significant differences among the liquid fertilizer-treated soils. The ASVs of the soil microbiomes clustered into 27 phyla of bacteria (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Ten of the phyla (\u003cem\u003eAcidobacteria, Actinobacteria, Bacteroidetes\u003c/em\u003e, \u003cem\u003eChloroflexi\u003c/em\u003e, \u003cem\u003eFirmicutes\u003c/em\u003e, \u003cem\u003eGemmatimonadetes\u003c/em\u003e, \u003cem\u003eNitrospirae\u003c/em\u003e, \u003cem\u003ePlanctomycetes\u003c/em\u003e, \u003cem\u003eProteobacteria\u003c/em\u003e, and \u003cem\u003eVerrucomicrobia\u003c/em\u003e) comprised over 1,000 of the ASVs detected from the soil bacteria in each sample; the ASVs of \u003cem\u003eActinobacteria\u003c/em\u003e and \u003cem\u003eProteobacteria\u003c/em\u003e comprised a larger percentage of soil bacterial DNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). A relatively small number of ASVs were found from \u003cem\u003eAcidobacteria\u003c/em\u003e and \u003cem\u003ePlanctomycetes\u003c/em\u003e in the CLF group and from \u003cem\u003eBacteroidetes\u003c/em\u003e, \u003cem\u003eGemmatimonadetes\u003c/em\u003e, and \u003cem\u003eNitrospirae\u003c/em\u003e in the non-treated group (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). Phyla accounting for a large amount of bacterial DNA were \u003cem\u003eFirmicutes\u003c/em\u003e in the untreated group and \u003cem\u003eVerrucomicrobia\u003c/em\u003e in the FLM and LBF groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo demonstrate the microbial changes induced by liquid fertilizers, we compared the significant differences (FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in ASVs of soil bacteria at the genus level (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Seventeen genera from seven bacterial phyla were increased in the CLF group compared to the control group; in particular, many CLF-induced genera were included in the phyla \u003cem\u003eActinobacteria\u003c/em\u003e (nine genera) and \u003cem\u003eProteobacteria\u003c/em\u003e (eight genera). \u003cem\u003eArthrobacter\u003c/em\u003e and \u003cem\u003eStreptomyces\u003c/em\u003e of the phylum \u003cem\u003eActinobacteria\u003c/em\u003e, \u003cem\u003eNeobacilus\u003c/em\u003e of the phylum \u003cem\u003eFirmicutes\u003c/em\u003e, and \u003cem\u003eLitorilinea\u003c/em\u003e of the phylum \u003cem\u003eChloroflexi\u003c/em\u003e were decreased by CLF compared to FLM and LBF. Twenty-three genus (14 genera increased by FLM and nine genera decreased by FLM) from six phyla were differentially distributed in the FLM group compared with the control group. Two genera (\u003cem\u003eLuteolibacter\u003c/em\u003e in the phylum \u003cem\u003eVerrucomicrobia\u003c/em\u003e and \u003cem\u003eActinoplanes\u003c/em\u003e in the phylum \u003cem\u003eActinobacteria\u003c/em\u003e) showed significant differences in bacterial composition between FLM and control groups. FLM increased the abundance of many genera of the phylum \u003cem\u003eProteobacteria\u003c/em\u003e, while \u003cem\u003ePriestia\u003c/em\u003e and \u003cem\u003eLitchfieldia\u003c/em\u003e of the phyla \u003cem\u003eFirmicutes\u003c/em\u003e, \u003cem\u003eNocardia, Nocardioides, Actinophytocola, Conexibacter, Pseudarthrobacter\u003c/em\u003e, and \u003cem\u003eAeromicrobium\u003c/em\u003e of the phylum \u003cem\u003eActinobacteria\u003c/em\u003e, and \u003cem\u003eSphingomonas\u003c/em\u003e of the phylum \u003cem\u003eProteobacteria\u003c/em\u003e were decreased in the FLM group. When comparing the LBF and control groups, \u003cem\u003eLuteolibacter\u003c/em\u003e (phylum \u003cem\u003eVerrucomicrobia\u003c/em\u003e) and \u003cem\u003eActinoplanes\u003c/em\u003e (phylum \u003cem\u003eActinobacteria\u003c/em\u003e) showed relatively large changes in bacterial composition, along with differences in distribution between FLM and control groups. Many bacterial genera that were increased by LBF from the phyla \u003cem\u003eActinobacteria\u003c/em\u003e (4 genera) and \u003cem\u003eProteobacteria\u003c/em\u003e (5 genera). \u003cem\u003eHydrogenispora\u003c/em\u003e of phylum \u003cem\u003eFirmicutes\u003c/em\u003e and \u003cem\u003eTrichocoleus\u003c/em\u003e of the phylum \u003cem\u003eCyanobacteria\u003c/em\u003e were decreased by LBF compared to control.\u003c/p\u003e \u003cp\u003eBacteria significantly correlated with chemical compositions\u003c/p\u003e \u003cp\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N, K, Na, and Ca from fertilizers, pH, and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N and exchangeable Na from soil were significantly correlated with the community of differentially distributed bacteria in the soils (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). We hypothesized that the seven chemical compositions of the fertilizers or soils influenced soil bacterial changes. Thus, we conducted a canonical correspondence analysis (CCA) between the seven chemical compositions and the differentially distributed bacteria (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) and evaluated the significance of the correlation (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Finally, we identified the significantly correlated genera among the five phyla. We found that six genera (\u003cem\u003eTerracoccus\u003c/em\u003e, \u003cem\u003eVirgibacillus\u003c/em\u003e, \u003cem\u003eLysobacter\u003c/em\u003e, \u003cem\u003eMesorhizobium\u003c/em\u003e, \u003cem\u003eNitrospira\u003c/em\u003e, and \u003cem\u003eRhizobium\u003c/em\u003e) were significantly correlated with the K content of the fertilizer. Interestingly, the six K-related genera had significant positive or negative correlations with one of the six chemical compositions, except for K. The CCA plot was divided into four quadrants (I-IV). The FLM group was located close to Na (soil) in quadrant I, while the LBF group was located close to NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N (fertilizer), Na (fertilizer), Ca (fertilizer), pH (soil), and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N (soil) in quadrant II (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). K (fertilizer) was correlated with the CLF group in quadrant IV. This suggests that the chemical composition of the fertilizers led to distinct bacterial communities.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSoil bacteria significantly correlated with chemical compositions determined by the Mantel test.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhylum\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGenus\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNa_F\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eK_F\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNO3_F\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCa_F\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003epH_S\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNO3_S\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNa_S\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003e\u003cem\u003eActinobacteria\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eActinoplanes\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.77*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.49\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eNocardioides\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-0.77*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-0.7*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-0.77*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-0.95***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e-0.72*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePedococcus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-0.7*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-0.84**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-0.81**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-0.94***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.7*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e-0.78*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePseudarthrobacter\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-0.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-0.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-0.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-0.81**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e-0.62\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTerrabacter\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-0.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-0.71*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-0.7*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-0.76*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e-0.78*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eTerracoccus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-0.85**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.75*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-0.9**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-0.93***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-0.81**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.89**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e-0.93***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eChloroflexi\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eLitorilinea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.86**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.75*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.88**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.8**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.94***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eFirmicutes\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eNeobacillus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-0.9***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.91***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.82**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.7*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.92***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.88**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eVirgibacillus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-0.81**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"7\" rowspan=\"8\"\u003e \u003cp\u003e\u003cem\u003eProteobacteria\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eLysobacter\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-0.78*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.83**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-0.94***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-0.92***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-0.73*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.94***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e-0.97***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eMassilia\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.81**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-0.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.78*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eMesorhizobium\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-0.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.74*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-0.69*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-0.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e-0.55\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eNitrosospira\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-0.72*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.74*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-0.9**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-0.85**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-0.89**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.79*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e-0.87**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eRhizobium\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-0.72*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.81**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-0.89**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-0.86**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-0.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.92***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e-0.9***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eRhizorhabdus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.79*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.78*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.83**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.71*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eSphingomonas\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-0.74*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-0.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-0.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-0.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-0.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e-0.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eSphingopyxis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-0.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-0.72*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-0.79*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e-0.69*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eVerrucomicrobia\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eLuteolibacter\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.76*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-0.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.78*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.74*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.75*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.75*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.59\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"9\"\u003eFold change value was calculated between treatments and control (log 2 [treatments / control]) by DESeq2 (NS, non-significance).\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"9\"\u003eThe significance of Pearson\u0026rsquo;s correlation coefficient (PCC) was determined by n-2 degrees of freedom (* \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, *** \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001)\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTo determine the specific distribution of bacteria with each fertilizer treatment, we compared the relative ASV abundance compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). \u003cem\u003eLitorilinea\u003c/em\u003e was decreased in the CLF group, but not in the FLM and LBF groups. The FLM group showed a specific decrease in \u003cem\u003eSphingomonas\u003c/em\u003e and \u003cem\u003eNocardioides\u003c/em\u003e. LBF showed increases in \u003cem\u003eSphingomonas\u003c/em\u003e and \u003cem\u003eNocardioides\u003c/em\u003e similar to CLF, and an increase in \u003cem\u003eLitorilinea\u003c/em\u003e similar to FLM, suggesting that the bacterial communities were in the middle of CLF and FLM.\u003c/p\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N and Na from the fertilizer and soil correlated with the FLM and LBF groups in this study. The ratio of nitrogen sources (NH\u003csub\u003e4\u003c/sub\u003e+-N or NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N) in chemical fertilizers led to distinct plant growth. Low ammonium (25%) and high nitrate (75%) have been shown to increase the plant growth of flowering Chinese cabbage (cv. Lvbao70, cv. Youlv80, and cv. Chixin No.2), while high ammonium (75%) and low nitrate (25%) had no significant effect on improving plant growth [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Furthermore, nitrate, in comparison with ammonium, has been shown to significantly increase the water content and leaf area of spinach, sunflower, and pea plants [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Although FLM and LBF had higher nitrate-nitrogen contents, the CLF group showed a stronger promotion of Chinese cabbage plant growth. We found higher amounts of Na in FLM and LBF liquid fertilizers and treated soils. In particular, the exchangeable Na in the liquid fertilizer was higher in FLM than in LBF. High levels of exchangeable Na in the soil have been shown to have negative effects on plant growth and soil nutrition [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. For example, exchangeable Na leads to reduced alfalfa and cotton growth according to its percentage in the soil [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The amount of exchangeable Na was shown to increase in the soil during a second cropping season using liquid fertilization developed from pig slurry compared to using chemical fertilizer [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Thus, we suggest that the high amount of exchangeable Na\u003csup\u003e+\u003c/sup\u003e in FLM and LBF inhibited plant development in Chinese cabbage.\u003c/p\u003e \u003cp\u003eThe organic fertilizers FLM and LBF led to increased levels of sugars, ascorbic acid, and antioxidants within the plants. Liquid organic fertilizer was found to increase the ascorbic acid and crude protein content in bell peppers (cv. Red Wonder F1) compared to mineral fertilizer [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Furthermore, the total content of phenolics, flavonoids, and glucosinolates in broccoli were increased by the additional application of organic fertilizers [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. \u003cem\u003eChlorella\u003c/em\u003e extracts have been shown to promote plant growth in pepper (\u003cem\u003eCapsicum annuum\u003c/em\u003e) plants [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. For example, plant height, leaf area, and fruit and shoot weights of pepper plants were increased after supplying \u003cem\u003eChlorella\u003c/em\u003e extracts for 21 days compared to the control. Furthermore, the antioxidant enzymes superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were increased with \u003cem\u003eChlorella\u003c/em\u003e extract treatment in pepper plants, suggesting the ability of \u003cem\u003eChlorella\u003c/em\u003e extracts to promote plant growth. On an organic farm, \u003cem\u003eChlorella\u003c/em\u003e culture treatments led to improved height, leaf width, fresh weight, and yield of Chinese chives and improved leaf thickness, leaf number, fresh weight, yield, and mineral content of spinach [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. \u003cem\u003eChlorella\u003c/em\u003e has economic value because of its diverse advantages such as plant growth promotion and functional food production in the application of plant bio-stimulants, including plant growth-promoting bacteria, microalgae, and mycorrhiza [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. \u003cem\u003eChlorella minutissima\u003c/em\u003e extracts had significantly higher DPPH RSA, FRAP, and iron chelating activity (ICA) values than those of other microalgal species (\u003cem\u003eDunaliella salina, Isochrysis galbana, Nannochloropsis oculate\u003c/em\u003e, and \u003cem\u003eTisochrysis lutea\u003c/em\u003e) [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. However, the effects of a \u003cem\u003eChlorella\u003c/em\u003e culture solution as a liquid bio-fertilizer on plant antioxidant activity have not yet been fully elucidated. We hypothesized that \u003cem\u003eChlorella\u003c/em\u003e culture liquid fertilizer could increase antioxidant levels in Chinese cabbage. The LBF group showed greater improvements in antioxidant levels compared to the CLF and FLM groups.\u003c/p\u003e \u003cp\u003eWe found that \u003cem\u003eLitorilinea\u003c/em\u003e was significantly decreased in the CLF group and increased in the FLM and LBF groups. \u003cem\u003eLitorilinea\u003c/em\u003e is an aerobic gram-negative bacterium [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Thus, it is possible that \u003cem\u003eLitorilinea\u003c/em\u003e increased due to anaerobic fermentation during the composting of the livestock manure used as a liquid fertilizer. \u003cem\u003eNocardioides\u003c/em\u003e is a genus of gram-positive aerobic bacteria belonging to the family Nocardioidaceae, whereas \u003cem\u003eSphingomonas\u003c/em\u003e is a genus of gram-negative aerobic bacteria belonging to the family Sphingomonadaceae [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Anaerobic fermentation for FLM production led to a decrease in \u003cem\u003eNocardioides\u003c/em\u003e and \u003cem\u003eSphingomonas\u003c/em\u003e. These bacteria contribute to nutrient cycling during the decomposition of complex biomasses and play a role in nitrogen fixation, which benefits plant growth and production [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. \u003cem\u003eSphingomonas\u003c/em\u003e can promote plant growth under normal and/or drought stress conditions owing to its contribution to root developmental plasticity [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. In the soil, a lack of \u003cem\u003eNocardioides\u003c/em\u003e and \u003cem\u003eSphingomonas\u003c/em\u003e may have led to the decreased growth rate of the FLM treated plants compared to the CLF and LBF groups. The newly developed LBF was generated by culturing \u003cem\u003eChlorella\u003c/em\u003e in an organic liquid fertilizer using livestock manure without a chemical medium. Recycling organic liquid fertilizers instead of chemical cultures is a cost-effective solution for the production of \u003cem\u003eChlorella\u003c/em\u003e biostimulants.\u003c/p\u003e"},{"header":"5 Conclusion","content":"\u003cp\u003eHere, we observed the effects of CLF and organic liquid fertilizers on plant growth, antioxidants, and soil microorganisms. To survey the changes of chemical components by different fertilizers, the amounts of NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N and Na were significantly different between the CLF and organic liquid fertilizers (FLM and LBF) groups. This result indicated that the plant growth of Chinese cabbage was inhibited in the FLM and LBF groups due to Na increase and the unsuitable ratio of NH\u003csub\u003e4\u003c/sub\u003e+-N and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N. However, LBF has improved the growth and substances of plants compared to FLM as an organic medium for \u003cem\u003eChlorella\u003c/em\u003e cultivation, suggesting the utilization of livestock manure. Finally, we have identified the distinct soil bacterial communities among different fertilizer-treated groups. For example, Litorilinea as anaerobic bacteria was decreased in the CLF group compared to FLM and LBF groups, which have needed anaerobic fermentation to produce livestock manure as raw material. Furthermore, Chinese cabbage may reduce plant growth in the FLM group by decreasing plant growth promoting bacterial species such as Nocardioides and Sphingomonas. Overall, the CLF affected to the highest growth rate of Chinese cabbage, while, the accumulation of plant substances was increased by organic liquid fertilizers (FLM and LBF). We identified that the \u003cem\u003eChlorella\u003c/em\u003e cultivation for LBF led to increases in plant growth and antioxidants. Thus, the development of organic bio-liquid fertilizers such as LBF will contribute to the construction of natural recycling systems through the application of organic fertilizers from livestock manure on organic farms.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCLF, chemical liquid fertilizer; FLM, fermented liquid Manure; LBF, liquid bio-fertilizer; PGPB, plant growth-promoting bacteria; EC, electrical conductivity; OM, organic matter; ASV, Amplicon Sequence Variant; NCBI, National Center for Biotechnology Information; FRAP, ferric reducing antioxidant power; CCA, canonical correspondence analysis; SOD, superoxide dismutase; POD, peroxidase; CAT, catalase; ICA, iron chelating activity;\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eOur research did not involve any studies with human or animal subjects. Experimental research on the plants complies with relevant institutional, national, and international guidelines and legislation and used for research and development.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry(IPET) and Korea Smart Farm R\u0026amp;D Foundation(KosFarm) through Smart Farm Innovation Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs(MAFRA) and Ministry of Science and ICT(MSIT), Rural Development Administration(RDA) (421046-03)\u003c/p\u003e\n\u003cp\u003eAvailability of data and materials\u003c/p\u003e\n\u003cp\u003eThe datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: BioProject, PRJNA982574\u003c/p\u003e\n\u003cp\u003eAuthor Contributions\u003c/p\u003e\n\u003cp\u003eJ.K.L. and S.G.H. conceived\u0026nbsp;of and designed the\u0026nbsp;study. J.K.L., N.Y.J., T.Y.L.L.,\u0026nbsp;W.Y.J., K.W.K., H.S.C., B.OL.,\u0026nbsp;S.R.K.,\u0026nbsp;and M.G.L. performed the field sampling.\u0026nbsp;J.K.L., N.Y.J., S.Y.S.,\u0026nbsp;S.R.K., M.G.L., and S.G.H. collected and analyzed the data. J.K.L.\u0026nbsp;and S.G.H. wrote the manuscript.\u0026nbsp;J.K.L. and N.Y.J. provided reagents and materials.\u0026nbsp;All authors contributed critically to the article, drafts, and revisions\u0026nbsp;and gave final approval for publication.\u003c/p\u003e\n\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eAuthor details\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eCollege of Life and Environment Science, Sangji University,\u0026nbsp;Wonju-si 26339,\u0026nbsp;Republic of Korea.\u0026nbsp;\u003csup\u003e2\u003c/sup\u003eDepartment\u0026nbsp;of Smart-Farm, Sangji University, Wonju-si\u0026nbsp;26339, Republic of\u0026nbsp;Korea.\u0026nbsp;\u003csup\u003e3\u003c/sup\u003eHoengseong Agricultural technology extension center, Hoengseong 25208,\u0026nbsp;Republic of\u0026nbsp;Korea.\u0026nbsp;\u003csup\u003e4\u003c/sup\u003eHanbio Incoporated. Hoengseong 25249,\u0026nbsp;Republic of\u0026nbsp;Korea.\u0026nbsp;\u003csup\u003e5\u003c/sup\u003eIndustry Academic Cooperation Foundation Resource Recycle Engineering Lab, Sangji University, Wonju-si\u0026nbsp;26339, Republic of Korea. \u003csup\u003e6\u003c/sup\u003eDepartment of Smart Life Science, Sangji University, Wonju-si 26339, Republic of Korea\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMaltas A, Charles R, Jeangros B, Sinaj S. Effect of organic fertilizers and reduced-tillage on soil properties, crop nitrogen response and crop yield: Results of a 12-year experiment in Changins, Switzerland. Soil Tillage Res. 2013;126:11\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTilman D. The greening of the green revolution. Nature. 1998;396(6708):211\u0026ndash;2.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiransari M. Soil microbes and plant fertilization. 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Microbiol Mol Biol Rev. 2017;81(2):e00063\u0026ndash;16.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLuo Y, Wang F, Huang Y, Zhou M, Gao J, Yan T, et al. Sphingomonas sp. Cra20 increases plant growth rate and alters rhizosphere microbial community structure of Arabidopsis thaliana under drought stress. Front Microbiol. 2019;10:1221.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePan F, Meng Q, Wang Q, Luo S, Chen B, Khan KY, et al. Endophytic bacterium Sphingomonas SaMR12 promotes cadmium accumulation by increasing glutathione biosynthesis in Sedum alfredii Hance. Chemosphere. 2016;154:358\u0026ndash;66.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhan AL, Waqas M, Kang S-M, Al-Harrasi A, Hussain J, Al-Rawahi A, et al. Bacterial endophyte Sphingomonas sp. LK11 produces gibberellins and IAA and promotes tomato plant growth. J Microbiol. 2014;52:689\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"organic fertilizer, bacterial community, bio-liquid fertilizer, Chinese cabbage, antioxidants","lastPublishedDoi":"10.21203/rs.3.rs-2964376/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2964376/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eEffluents from livestock excretion have worldwide environmental disadvantages, such as air and water pollution. However, livestock manure and organic liquid fertilizers developed for the proper management of livestock excretions can be used as environmentally friendly fertilizers for sustainable agriculture. Therefore, we investigated the effects of organic liquid fertilizers on the growth and antioxidant accumulation in Chinese cabbage (\u003cem\u003eBrassica rapa\u003c/em\u003e subsp. \u003cem\u003ePekinensis\u003c/em\u003e).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThree liquid fertilizers, chemical liquid fertilizer (CLF), fermented liquid manure (FLM) from pig droppings, and liquid bio-fertilizer (LBF) from \u003cem\u003eChlorella\u003c/em\u003e cultured in purified organic liquid manure, were used in this experiment. Plant performance was observed and soil microbial changes caused by these liquid fertilizers were analyzed using 16S rRNA sequencing. We observed the highest plant growth in terms of plant length, chlorophyll concentration, width and length of leaves, and fresh and dry weights in the CLF group. LBF led to improved plant growth compared to FLM. Chlorophyll concentrations and color measurements of the plants were higher with LBF than with FLM. Interestingly, LBF affected plant substances with respect to sugar, ascorbic acid, and antioxidants in Chinese cabbage compared to CLF and FLM. The highest total polyphenol and flavonoid content, antioxidant activity, nitrite-scavenging capacity, and reducing power were observed in the LBF group. Significant changes in the bacterial population were observed in amplicon sequence variant analysis; the presence of Verrucomicrobia increased in soils after FLM and LBF treatments. LBF-treated soils had a higher abundance of Proteobacteria than FLM-treated soils.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eWe studied the effects of organic fertilizers across different liquid fertilizers on plant growth, antioxidants, and soil microorganisms. Especially, the LBF as organic bio-liquid fertilizer improved the plant growth and substances in Chinese cabbage under a controlled environment agriculture system. We have identified the specific bacterial species regarding the production process of liquid fertilizers in the change of soil microbial communities by the short-term experimental treatment. Thus, the proper development of organic bio-liquid fertilizer can contribute to the organic waste-recycling systems of livestock excretions for sustainable agriculture.\u003c/p\u003e","manuscriptTitle":"Influence of organic liquid fertilizer developed from livestock manure on the growth, antioxidant activities, and soil microbial populations of Chinese cabbage","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-06-16 14:45:30","doi":"10.21203/rs.3.rs-2964376/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"dd31a72c-1ea0-430f-95fc-ed0f12efe1ec","owner":[],"postedDate":"June 16th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-03-31T23:53:16+00:00","versionOfRecord":[],"versionCreatedAt":"2023-06-16 14:45:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2964376","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2964376","identity":"rs-2964376","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}