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Identification and characterization of proteins, lipids, and metabolites in two organic fertilizer products derived from different nutrient sources

Identification and characterization of proteins, lipids, and metabolites in two organic... The biochemical composition of organic fertilizers largely determines their nutrient supply characteristics follow- ing soil application as well as their potential impact on soil microbial communities. Yet, limited information is avail- able regarding the biochemical composition of organic fertilizers derived from different nutrient sources. Here, we qualitatively analyzed the presence and abundance of proteins, lipids, and metabolites in a liquid fish fertilizer (LFF) product and a type of granular organic fertilizer (GOF) commonly used in organic vegetable production, using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Our results suggest that the presence and abundance of proteins, lipids, and metabolites differ greatly between GOF and LFF. The qualitative analysis shows LFF as a rich source of metabolites, while complex proteins and long-chain saturated fatty acids are dominant in GOF. The degree of biochemical composition complexity may help explain the varying impacts of different types of organic fertilizers on nutrient availability, soil health, and environmental quality. Keywords: Biochemical composition, LC–MS/MS, Liquid chromatography–tandem mass spectrometry, Lipidomics, Metabolomics, Nutrient availability, Organic nutrient source, Proteomics Introduction composition through addition of C and nitrogen (N)-rich Organic fertilizers derived from various animal and organic compounds [3–6]. At the same time, the nutrient plant-based byproducts have been widely used as nutri- release process of organic fertilizers relies substantially ent sources for organic vegetable crop production. Com- on the complex biochemical transformation activities pared to synthetic chemical fertilizers, organic fertilizers mediated by soil microbes [7] in addition to environmen- tend to have lower nutrient content along with variability tal conditions. in composition that depends on ingredients. However, Early studies indicated that the general C/N ratio and organic fertilizers often contain beneficial microorgan - nutrient analysis of organic fertilizers might not  offer isms and are generally rich in organic carbon (C) [1]. sufficient information for predicting nutrient release Applications of organic fertilizers have been shown to patterns, while the biochemical composition of organic increase soil organic matter content, enhance overall soil fertilizers could be highly associated with mineralization enzyme activity [2], and influence microbial community kinetics [8, 9]. Numerous methods have been developed to estimate N availability in soils after organic fertilizer application, such as incubation-based N mineraliza- *Correspondence: zxin@ufl.edu tion studies [10, 11]. Most recently, the extractable soil Horticultural Sciences Department, University of Florida, Gainesville, FL protein pool was suggested to be a soil health indicator 32611-0690, USA of potentially available organic N [12]. Plant metabolite Full list of author information is available at the end of the article © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Li et al. Appl Biol Chem (2021) 64:72 Page 2 of 6 compounds also influence soil nutrient cycling, such as acidified mobile phases of water and acetonitrile for C and N mineralization, through their impact on soil metabolites and water, acetonitrile, and isopropanol for organisms [13]. The input of lipids, especially the long- lipids. Proteins (10 µg) were separated on a PepMAP col- chain compounds, play an important role in soil health umn using acidified water and acetonitrile. enhancement including the accumulation of soil organic Both the lipid and metabolite compounds were ana- matter content [14]. Liquid chromatography–tandem lyzed on a Bruker Impact II QqTOF mass spectrometer mass spectrometry (LC–MS/MS) with high-throughput (Bruker Daltonics, Billerica, MA, USA) using electro- capacities has been demonstrated to be a sensitive and spray ionization operated in positive mode. Proteins were powerful analytical tool for detecting proteins, lipids, and analyzed on a Thermo Scientific Q Exactive HF Orbitrap other metabolites from biological materials of various mass spectrometer  equipped with an EASY Nanospray sources including soil [15] and plant tissue samples [16– source operated in positive mode. All employed data 18]. However, limited information is available regarding dependent collisionally-activated dissociation. Proteom- specific biochemical compounds contained in organic ics data were analyzed in Proteome Discoverer (version fertilizers that may affect soil microbial communities and 2.4) using the SEQUEST HT searching algorithm. Lipids nutrient availability as well as environmental quality. LC– and metabolites were analyzed in MetaboScape (version MS/MS has rarely been used to compare biochemical 4.0). Full experimental details are provided in Additional compositions of organic fertilizers with different nutri - file 1. ent sources. Here, we chose to examine two commer- cially available organic fertilizer products, a liquid fish Results and discussion fertilizer  (LFF) derived from enzymatically digested fish Proteomics proteins and a granular organic fertilizer (GOF) contain- Figure 1B is a graphical summary of total spectral counts ing feather meal and other animal waste materials, which for each protein detected in   GOF and   LFF products. represent commonly used nutrient sources for organic Hemoglobin (632 spectral counts), keratin (83 spectral crop production [19]. counts), and actin (47 spectral counts) were the major Therefore, the objective of this pilot study was to proteins detected in GOF (862 spectral counts in total). employ LC–MS/MS to identify and characterize pro- In contrast, parvalbumin (28 spectral counts), tropomy- teins, lipids, and metabolites in two commonly used osin (23 spectral counts), and actin (11 spectral counts) commercial organic fertilizer products for qualitative were abundant in LFF (96 spectral counts in total). Deg- comparisons of their biochemical characteristics. radation of proteins in the soil can be affected by their intrinsic structures including amyloid fibril formation Materials and methods and glycosylation, accessibility to soil microbes or extra- The LFF  analyzed in this study can be used in organic cellular enzymes, and complex structure formation with fertigation systems through drip irrigation, while the other soil organic compounds such as tannins, lignin, GOF  may be used for preplant application and/or sid- and humic substances [21]. For instance, hemoglobin edressing during the crop production season. Both can bind tannins to form protein–polyphenol complex organic fertilizer products have higher levels of N than (PPC) that limits N mineralization due to its resistance to phosphorus (P) and potassium (K) (Figure 1A; Additional decomposition [22]. As a fibrous structural protein, kera - file  1: Table S1). Equal amounts of LFF (100 µL) and GOF tin could be highly recalcitrant in response to microbial (100  mg) were individually extracted for proteins, lipids, degradation because of its molecular architecture that and metabolites. The equivalent mass of 100 µL LFF was involves formation of disulfide bonds, phosphorylation, determined as 97.5 ± 1.0 mg. Lipids were extracted via a and glycosylation [23]. The predominance of hemoglobin modified Folch method [20], metabolites were extracted and keratin found in GOF might imply lower N minerali- by ice-cold methanol, and proteins by acetone/metha- zation rates of GOF relative to LFF. nol precipitation. The protein pellet was reconstituted in 0.2% surfactant enhancer and concentrations were meas- Lipidomics ured via a Qubit Fluorometer (Thermo Fisher Scientific Phosphatidylcholine (PC) (e.g., C42, C44, C46) and Inc., Waltham, MA, USA). In-gel protein digestion was diglyceride (DAG) (e.g., C31, C35, C37, C39, C41, C43, performed using trypsin as the enzyme (Promega Corpo- C47) lipids accounted for 39.4% and 26.0% of the lipids ration, Madison, WI, USA). LC analysis for all was per- in GOF, respectively, whereas DAGs (e.g., C33, C35, C37, formed on a Dionex UltiMate 3000 RSLCnano system C39, C41, C43, C47) showed a higher classification pro - (Thermo Fisher Scientific Inc.). Lipid and metabolites portion (53.9%) in LFF (Figure 2). Our data also revealed were separately injected (5 µL) on an Acclaim PepMap that the majority of DAGs in both LFF and GOF con- RSLC C18 column (Thermo Fisher Scientific Inc.) using tained highly polyunsaturated fatty acids (e.g., 30:4, 32:3, Li  et al. Appl Biol Chem (2021) 64:72 Page 3 of 6 Figure 1 A Visual appearance of the two types of organic fertilizer products analyzed. Left: liquid fish fertilizer (LFF); Right: granular organic fertilizer (GOF). B Total spectral counts for each protein identified in GOF and LFF. G3PD: glyceraldehyde-3-phosphate dehydrogenase, IgH: immunoglobulin heavy chain, MZMP: mitochondrial zinc maintenance protein, PGM: phosphoglucosamine mutase, tRNA_PS: tRNA pseudouridine synthase. Y-axis scale is made disproportionally to the spectral count in order to capture the wide range of spectral counts of all proteins identified and indicate the actual values of spectral counts for each protein. 34:5, 40:9, 44:12), which could be more susceptible to identified in GOF may degrade and release essential ele - microbial degradation in the soil than saturated fatty acid ments rather slowly due to its long-chain structure and molecules [24]. Conversely, PC lipids containing N and P the relatively high degree of fatty acid saturation. Overall, Li et al. Appl Biol Chem (2021) 64:72 Page 4 of 6 Category Class Neutral glycoshingolipids Phosphatidylcholine Phosphatidylserine Phosphosphingolipids Sterols Phosphatidylethanolamine Oxidized glycerophospholipids Ceramides Diglyceride 0.8% Triglyceride Glycerophosphocholine 66.3% Acidic glycosphingolipids 4.9% 1.8% 1.5% Sterol lipids Sphingolipids Glycerolipids Glycerophospholipids Figure 2 Different categories and classes of lipids identified in granular organic fertilizer (GOF) and liquid fish fertilizer (LFF). The interior donut represents LFF, and the exterior donut represents GOF. The area of each color represents the relative abundance (%) indicated. Based on the Lipid Metabolites and Pathways Strategy (LIPID MAPS) classification system, lipids are divided into four categories: sterol lipids, sphingolipids, glycerolipids, and glycerophospholipids. Each category is further divided into classes. Glycerolipids: diglyceride and triglyceride; Glycerophospholipids: phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, glycerophosphocholine, and oxidized glycerophospholipids; Sterol lipids: sterols; Sphingolipids: phosphosphingolipids, neutral glycosphingolipids, ceramides, and acidic glycosphingolipids. the diverse array of lipids identified in organic fertiliz - property of capsaicin [27]. Four fatty acid amides includ- ers in our study might indicate the potential impact of ing pipericine (m/z 336.3260; RT 40.04  min), macamide organic fertilizer application on improving soil health. (m/z 346.3100; RT 37.08  min), docosanamide (m/z For instance, lipids may serve as C and energy sources 340.3572; RT 42.44  min), and erucamide (m/z 338.3416; for various lipid-degrading soil microorganisms, such RT 41.87 min) appeared more abundant in LFF than GOF as Bacillus, Arthrobacter, and Pseudomonas [25]. Long- (Table S2), but statistical analyses with replications were chain lipids (> C20), in particular, are important in C not performed in this exploratory study. These amides stabilization and humification processes during the accu - may participate in stimulatory activities associated with mulation of soil organic matter [26]. soil microbial metabolism [28]. Two phenols, including p-coumaric acid ethyl ester (m/z 193.0861; RT 35.01 min) Metabolomics and gingerol (m/z 277.1798; RT 19.20  min), were also The identified metabolites (Additional file  1: Table  S2) abundant in LFF (Table  S2). As a product of acidic were categorized as amines, amides, polyols, organic hydrolysis of p-coumaric acid ethyl ester, p-coumaric acid acids, steroids, vitamins, isoprenoids, and plasticizers can increase soil dehydrogenase activity and abundance (Figure  3). Comparisons between LFF and GOF sug- of soil bacterial and fungal communities [29, 30]. How- gested that LFF is a richer source of metabolites (Fig- ever, gingerol, another compound abundant in LFF, may ure 3) and the two organic fertilizers might have different display antimicrobial activity [31]. Additionally, given the impacts on soil nutrient cycling and soil microbial com- relatively high acidity (pH = 3.5) and abundant level of munities. For example, stearamide (m/z 284.2949; reten- organic acids of LFF (Additional file  1: Tables S1, S2), its tion time (RT) 36.74  min) was detected in LFF but not application could potentially result in a reduction in soil in GOF. It is interesting that capsaicin (m/z 308.2218; RT pH and suppression of certain soilborne pathogens in the 15.05  min) was found in GOF but absent in LFF (Addi- longer term [32]. The low pH of LFF may be attributed tional file  1: Table  S2). Although the source of capsaicin to the abundance of bile acids, such as 3β-hydroxy-5- is unclear, the GOF that contains capsaicin might poten- cholenoic acid (m/z 357.2787; RT 22.11  min), 3-oxo- tially demonstrate a deterrent or repellent effect on cer - cholic acid (m/z 424.3060; RT 17.59  min), cholic acid tain fungi, insects, and mammals due to the irritant (m/z 426.3214; RT 18.98 min), and nutriacholic acid (m/z Li  et al. Appl Biol Chem (2021) 64:72 Page 5 of 6 derived from various nutrient sources and include quanti- tative analysis for in-depth comparisons. Overall, our study demonstrates the complexity in biochemical composi- tion of organic fertilizers and suggests the need to further understand how organic fertilizers with different biochemi - cal profiles influence nutrient cycling, soil health, and envi - Amines ronmental quality. Amides Polyols Organic acids 600 Isoprenoids Intensity x 10 Abbreviations Steroids LFF: Liquid fish fertilizer; GOF: Granular organic fertilizer; GC/MS: Gas chroma- Vitamins Plasticizers tography–mass spectrometry; LC–MS/MS: Liquid chromatography–tandem mass spectrometry; PPC: Protein–polyphenol complex; PC: Phosphatidylcho- line; DAG: Diglyceride. Supplementary Information The online version contains supplementary material available at https:// doi. org/ 10. 1186/ s13765- 021- 00625-2. 10 20 30 40 50 60 Additional file 1: Table S1. Basic characteristics of the two organic Retention time (min) fertilizer products used in this study. Table S2. List of metabolites Figure 3 Survey overview of metabolite presence and relative identified by LC-MS/MS in liquid fish fertilizer and granular organic abundance broken down by class and functionality for liquid fertilizer. fish fertilizer (A) and granular organic fertilizer (B). These compounds were annotated and categorized with the help of PubChem based on the general chemical structures of compounds. Acknowledgements This work was supported by Organic Agriculture Research and Extension Ini- The compound abundance was determined using the spectral and tiative grant no. 2015-51300-24134 from the USDA National Institute of Food analyte metabolite matches compiled in MetaboScape. The circle and Agriculture. We thank James Colee for his assistance with data analyses. color represents compound category and circle size is proportional We also thank the Mass Spectrometry Research and Education Center at the to the compound abundance. The abundance values were scaled up University of Florida and their funding source: NIH S10 OD021758-01A1. by an intensity factor of 10 in plot B in order to make dots visible in plot B. Authors’ contributions JL and XZ designed the experiment and wrote the manuscript. LSB, MNK, and KBB performed the LC–MS/MS analysis and helped with data analysis and interpretation. All authors read and approved the final manuscript. 391.2844; RT 20.55  min) (Table  S2). Moreover, bacterial Funding degradation products (e.g., androstadienediones) of bile Funding was received from the Organic Agriculture Research and Extension acids could also pose   a potential risk, such as reduced Initiative (Grant No. 2015-51300-24134) funded by the USDA National Institute of Food and Agriculture. reproduction rates, to invertebrates in agricultural soils [33]. Similarly, as a steroid hormone, progesterone (m/z Availability of data and materials 315.2319; RT 28.67  min), which was discovered in both All data generated or analyzed during the present study are included in this published article. LFF and GOF (Additional file  1: Table  S2), might  accu- mulate in soil and cause adverse impacts on the Declarations environment [34]. Competing interests There is no conflict of interest. Conclusions Our qualitative analysis is the first study attempting to elu - Author details cidate the biochemical composition of organic fertilizers, Horticultural Sciences Department, University of Florida, Gainesville, FL 32611-0690, USA. Department of Chemistry, University of Florida, Gainesville, explore its linkage to nutrient availability of organic ferti- FL 32611-7200, USA. lizers, and envision its impact on soil quality and health. The dominance of complex proteins and long-chain satu - Received: 13 April 2021 Accepted: 13 July 2021 rated fatty acids contained in GOF suggests that GOF might decompose and release nutrients at a slower rate in the soil  relative to LFF. A diverse variety and abundance References of metabolites were identified in GOF and LFF, indicating 1. 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Identification and characterization of proteins, lipids, and metabolites in two organic fertilizer products derived from different nutrient sources

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Abstract

The biochemical composition of organic fertilizers largely determines their nutrient supply characteristics follow- ing soil application as well as their potential impact on soil microbial communities. Yet, limited information is avail- able regarding the biochemical composition of organic fertilizers derived from different nutrient sources. Here, we qualitatively analyzed the presence and abundance of proteins, lipids, and metabolites in a liquid fish fertilizer (LFF) product and a type of granular organic fertilizer (GOF) commonly used in organic vegetable production, using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Our results suggest that the presence and abundance of proteins, lipids, and metabolites differ greatly between GOF and LFF. The qualitative analysis shows LFF as a rich source of metabolites, while complex proteins and long-chain saturated fatty acids are dominant in GOF. The degree of biochemical composition complexity may help explain the varying impacts of different types of organic fertilizers on nutrient availability, soil health, and environmental quality. Keywords: Biochemical composition, LC–MS/MS, Liquid chromatography–tandem mass spectrometry, Lipidomics, Metabolomics, Nutrient availability, Organic nutrient source, Proteomics Introduction composition through addition of C and nitrogen (N)-rich Organic fertilizers derived from various animal and organic compounds [3–6]. At the same time, the nutrient plant-based byproducts have been widely used as nutri- release process of organic fertilizers relies substantially ent sources for organic vegetable crop production. Com- on the complex biochemical transformation activities pared to synthetic chemical fertilizers, organic fertilizers mediated by soil microbes [7] in addition to environmen- tend to have lower nutrient content along with variability tal conditions. in composition that depends on ingredients. However, Early studies indicated that the general C/N ratio and organic fertilizers often contain beneficial microorgan - nutrient analysis of organic fertilizers might not  offer isms and are generally rich in organic carbon (C) [1]. sufficient information for predicting nutrient release Applications of organic fertilizers have been shown to patterns, while the biochemical composition of organic increase soil organic matter content, enhance overall soil fertilizers could be highly associated with mineralization enzyme activity [2], and influence microbial community kinetics [8, 9]. Numerous methods have been developed to estimate N availability in soils after organic fertilizer application, such as incubation-based N mineraliza- *Correspondence: zxin@ufl.edu tion studies [10, 11]. Most recently, the extractable soil Horticultural Sciences Department, University of Florida, Gainesville, FL protein pool was suggested to be a soil health indicator 32611-0690, USA of potentially available organic N [12]. Plant metabolite Full list of author information is available at the end of the article © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Li et al. Appl Biol Chem (2021) 64:72 Page 2 of 6 compounds also influence soil nutrient cycling, such as acidified mobile phases of water and acetonitrile for C and N mineralization, through their impact on soil metabolites and water, acetonitrile, and isopropanol for organisms [13]. The input of lipids, especially the long- lipids. Proteins (10 µg) were separated on a PepMAP col- chain compounds, play an important role in soil health umn using acidified water and acetonitrile. enhancement including the accumulation of soil organic Both the lipid and metabolite compounds were ana- matter content [14]. Liquid chromatography–tandem lyzed on a Bruker Impact II QqTOF mass spectrometer mass spectrometry (LC–MS/MS) with high-throughput (Bruker Daltonics, Billerica, MA, USA) using electro- capacities has been demonstrated to be a sensitive and spray ionization operated in positive mode. Proteins were powerful analytical tool for detecting proteins, lipids, and analyzed on a Thermo Scientific Q Exactive HF Orbitrap other metabolites from biological materials of various mass spectrometer  equipped with an EASY Nanospray sources including soil [15] and plant tissue samples [16– source operated in positive mode. All employed data 18]. However, limited information is available regarding dependent collisionally-activated dissociation. Proteom- specific biochemical compounds contained in organic ics data were analyzed in Proteome Discoverer (version fertilizers that may affect soil microbial communities and 2.4) using the SEQUEST HT searching algorithm. Lipids nutrient availability as well as environmental quality. LC– and metabolites were analyzed in MetaboScape (version MS/MS has rarely been used to compare biochemical 4.0). Full experimental details are provided in Additional compositions of organic fertilizers with different nutri - file 1. ent sources. Here, we chose to examine two commer- cially available organic fertilizer products, a liquid fish Results and discussion fertilizer  (LFF) derived from enzymatically digested fish Proteomics proteins and a granular organic fertilizer (GOF) contain- Figure 1B is a graphical summary of total spectral counts ing feather meal and other animal waste materials, which for each protein detected in   GOF and   LFF products. represent commonly used nutrient sources for organic Hemoglobin (632 spectral counts), keratin (83 spectral crop production [19]. counts), and actin (47 spectral counts) were the major Therefore, the objective of this pilot study was to proteins detected in GOF (862 spectral counts in total). employ LC–MS/MS to identify and characterize pro- In contrast, parvalbumin (28 spectral counts), tropomy- teins, lipids, and metabolites in two commonly used osin (23 spectral counts), and actin (11 spectral counts) commercial organic fertilizer products for qualitative were abundant in LFF (96 spectral counts in total). Deg- comparisons of their biochemical characteristics. radation of proteins in the soil can be affected by their intrinsic structures including amyloid fibril formation Materials and methods and glycosylation, accessibility to soil microbes or extra- The LFF  analyzed in this study can be used in organic cellular enzymes, and complex structure formation with fertigation systems through drip irrigation, while the other soil organic compounds such as tannins, lignin, GOF  may be used for preplant application and/or sid- and humic substances [21]. For instance, hemoglobin edressing during the crop production season. Both can bind tannins to form protein–polyphenol complex organic fertilizer products have higher levels of N than (PPC) that limits N mineralization due to its resistance to phosphorus (P) and potassium (K) (Figure 1A; Additional decomposition [22]. As a fibrous structural protein, kera - file  1: Table S1). Equal amounts of LFF (100 µL) and GOF tin could be highly recalcitrant in response to microbial (100  mg) were individually extracted for proteins, lipids, degradation because of its molecular architecture that and metabolites. The equivalent mass of 100 µL LFF was involves formation of disulfide bonds, phosphorylation, determined as 97.5 ± 1.0 mg. Lipids were extracted via a and glycosylation [23]. The predominance of hemoglobin modified Folch method [20], metabolites were extracted and keratin found in GOF might imply lower N minerali- by ice-cold methanol, and proteins by acetone/metha- zation rates of GOF relative to LFF. nol precipitation. The protein pellet was reconstituted in 0.2% surfactant enhancer and concentrations were meas- Lipidomics ured via a Qubit Fluorometer (Thermo Fisher Scientific Phosphatidylcholine (PC) (e.g., C42, C44, C46) and Inc., Waltham, MA, USA). In-gel protein digestion was diglyceride (DAG) (e.g., C31, C35, C37, C39, C41, C43, performed using trypsin as the enzyme (Promega Corpo- C47) lipids accounted for 39.4% and 26.0% of the lipids ration, Madison, WI, USA). LC analysis for all was per- in GOF, respectively, whereas DAGs (e.g., C33, C35, C37, formed on a Dionex UltiMate 3000 RSLCnano system C39, C41, C43, C47) showed a higher classification pro - (Thermo Fisher Scientific Inc.). Lipid and metabolites portion (53.9%) in LFF (Figure 2). Our data also revealed were separately injected (5 µL) on an Acclaim PepMap that the majority of DAGs in both LFF and GOF con- RSLC C18 column (Thermo Fisher Scientific Inc.) using tained highly polyunsaturated fatty acids (e.g., 30:4, 32:3, Li  et al. Appl Biol Chem (2021) 64:72 Page 3 of 6 Figure 1 A Visual appearance of the two types of organic fertilizer products analyzed. Left: liquid fish fertilizer (LFF); Right: granular organic fertilizer (GOF). B Total spectral counts for each protein identified in GOF and LFF. G3PD: glyceraldehyde-3-phosphate dehydrogenase, IgH: immunoglobulin heavy chain, MZMP: mitochondrial zinc maintenance protein, PGM: phosphoglucosamine mutase, tRNA_PS: tRNA pseudouridine synthase. Y-axis scale is made disproportionally to the spectral count in order to capture the wide range of spectral counts of all proteins identified and indicate the actual values of spectral counts for each protein. 34:5, 40:9, 44:12), which could be more susceptible to identified in GOF may degrade and release essential ele - microbial degradation in the soil than saturated fatty acid ments rather slowly due to its long-chain structure and molecules [24]. Conversely, PC lipids containing N and P the relatively high degree of fatty acid saturation. Overall, Li et al. Appl Biol Chem (2021) 64:72 Page 4 of 6 Category Class Neutral glycoshingolipids Phosphatidylcholine Phosphatidylserine Phosphosphingolipids Sterols Phosphatidylethanolamine Oxidized glycerophospholipids Ceramides Diglyceride 0.8% Triglyceride Glycerophosphocholine 66.3% Acidic glycosphingolipids 4.9% 1.8% 1.5% Sterol lipids Sphingolipids Glycerolipids Glycerophospholipids Figure 2 Different categories and classes of lipids identified in granular organic fertilizer (GOF) and liquid fish fertilizer (LFF). The interior donut represents LFF, and the exterior donut represents GOF. The area of each color represents the relative abundance (%) indicated. Based on the Lipid Metabolites and Pathways Strategy (LIPID MAPS) classification system, lipids are divided into four categories: sterol lipids, sphingolipids, glycerolipids, and glycerophospholipids. Each category is further divided into classes. Glycerolipids: diglyceride and triglyceride; Glycerophospholipids: phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, glycerophosphocholine, and oxidized glycerophospholipids; Sterol lipids: sterols; Sphingolipids: phosphosphingolipids, neutral glycosphingolipids, ceramides, and acidic glycosphingolipids. the diverse array of lipids identified in organic fertiliz - property of capsaicin [27]. Four fatty acid amides includ- ers in our study might indicate the potential impact of ing pipericine (m/z 336.3260; RT 40.04  min), macamide organic fertilizer application on improving soil health. (m/z 346.3100; RT 37.08  min), docosanamide (m/z For instance, lipids may serve as C and energy sources 340.3572; RT 42.44  min), and erucamide (m/z 338.3416; for various lipid-degrading soil microorganisms, such RT 41.87 min) appeared more abundant in LFF than GOF as Bacillus, Arthrobacter, and Pseudomonas [25]. Long- (Table S2), but statistical analyses with replications were chain lipids (> C20), in particular, are important in C not performed in this exploratory study. These amides stabilization and humification processes during the accu - may participate in stimulatory activities associated with mulation of soil organic matter [26]. soil microbial metabolism [28]. Two phenols, including p-coumaric acid ethyl ester (m/z 193.0861; RT 35.01 min) Metabolomics and gingerol (m/z 277.1798; RT 19.20  min), were also The identified metabolites (Additional file  1: Table  S2) abundant in LFF (Table  S2). As a product of acidic were categorized as amines, amides, polyols, organic hydrolysis of p-coumaric acid ethyl ester, p-coumaric acid acids, steroids, vitamins, isoprenoids, and plasticizers can increase soil dehydrogenase activity and abundance (Figure  3). Comparisons between LFF and GOF sug- of soil bacterial and fungal communities [29, 30]. How- gested that LFF is a richer source of metabolites (Fig- ever, gingerol, another compound abundant in LFF, may ure 3) and the two organic fertilizers might have different display antimicrobial activity [31]. Additionally, given the impacts on soil nutrient cycling and soil microbial com- relatively high acidity (pH = 3.5) and abundant level of munities. For example, stearamide (m/z 284.2949; reten- organic acids of LFF (Additional file  1: Tables S1, S2), its tion time (RT) 36.74  min) was detected in LFF but not application could potentially result in a reduction in soil in GOF. It is interesting that capsaicin (m/z 308.2218; RT pH and suppression of certain soilborne pathogens in the 15.05  min) was found in GOF but absent in LFF (Addi- longer term [32]. The low pH of LFF may be attributed tional file  1: Table  S2). Although the source of capsaicin to the abundance of bile acids, such as 3β-hydroxy-5- is unclear, the GOF that contains capsaicin might poten- cholenoic acid (m/z 357.2787; RT 22.11  min), 3-oxo- tially demonstrate a deterrent or repellent effect on cer - cholic acid (m/z 424.3060; RT 17.59  min), cholic acid tain fungi, insects, and mammals due to the irritant (m/z 426.3214; RT 18.98 min), and nutriacholic acid (m/z Li  et al. Appl Biol Chem (2021) 64:72 Page 5 of 6 derived from various nutrient sources and include quanti- tative analysis for in-depth comparisons. Overall, our study demonstrates the complexity in biochemical composi- tion of organic fertilizers and suggests the need to further understand how organic fertilizers with different biochemi - cal profiles influence nutrient cycling, soil health, and envi - Amines ronmental quality. Amides Polyols Organic acids 600 Isoprenoids Intensity x 10 Abbreviations Steroids LFF: Liquid fish fertilizer; GOF: Granular organic fertilizer; GC/MS: Gas chroma- Vitamins Plasticizers tography–mass spectrometry; LC–MS/MS: Liquid chromatography–tandem mass spectrometry; PPC: Protein–polyphenol complex; PC: Phosphatidylcho- line; DAG: Diglyceride. Supplementary Information The online version contains supplementary material available at https:// doi. org/ 10. 1186/ s13765- 021- 00625-2. 10 20 30 40 50 60 Additional file 1: Table S1. Basic characteristics of the two organic Retention time (min) fertilizer products used in this study. Table S2. List of metabolites Figure 3 Survey overview of metabolite presence and relative identified by LC-MS/MS in liquid fish fertilizer and granular organic abundance broken down by class and functionality for liquid fertilizer. fish fertilizer (A) and granular organic fertilizer (B). These compounds were annotated and categorized with the help of PubChem based on the general chemical structures of compounds. Acknowledgements This work was supported by Organic Agriculture Research and Extension Ini- The compound abundance was determined using the spectral and tiative grant no. 2015-51300-24134 from the USDA National Institute of Food analyte metabolite matches compiled in MetaboScape. The circle and Agriculture. We thank James Colee for his assistance with data analyses. color represents compound category and circle size is proportional We also thank the Mass Spectrometry Research and Education Center at the to the compound abundance. The abundance values were scaled up University of Florida and their funding source: NIH S10 OD021758-01A1. by an intensity factor of 10 in plot B in order to make dots visible in plot B. Authors’ contributions JL and XZ designed the experiment and wrote the manuscript. LSB, MNK, and KBB performed the LC–MS/MS analysis and helped with data analysis and interpretation. All authors read and approved the final manuscript. 391.2844; RT 20.55  min) (Table  S2). Moreover, bacterial Funding degradation products (e.g., androstadienediones) of bile Funding was received from the Organic Agriculture Research and Extension acids could also pose   a potential risk, such as reduced Initiative (Grant No. 2015-51300-24134) funded by the USDA National Institute of Food and Agriculture. reproduction rates, to invertebrates in agricultural soils [33]. Similarly, as a steroid hormone, progesterone (m/z Availability of data and materials 315.2319; RT 28.67  min), which was discovered in both All data generated or analyzed during the present study are included in this published article. LFF and GOF (Additional file  1: Table  S2), might  accu- mulate in soil and cause adverse impacts on the Declarations environment [34]. Competing interests There is no conflict of interest. Conclusions Our qualitative analysis is the first study attempting to elu - Author details cidate the biochemical composition of organic fertilizers, Horticultural Sciences Department, University of Florida, Gainesville, FL 32611-0690, USA. Department of Chemistry, University of Florida, Gainesville, explore its linkage to nutrient availability of organic ferti- FL 32611-7200, USA. lizers, and envision its impact on soil quality and health. The dominance of complex proteins and long-chain satu - Received: 13 April 2021 Accepted: 13 July 2021 rated fatty acids contained in GOF suggests that GOF might decompose and release nutrients at a slower rate in the soil  relative to LFF. A diverse variety and abundance References of metabolites were identified in GOF and LFF, indicating 1. 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Journal

Applied Biological ChemistrySpringer Journals

Published: Dec 1, 2021

Keywords: Biochemical composition; LC–MS/MS; Liquid chromatography–tandem mass spectrometry; Lipidomics; Metabolomics; Nutrient availability; Organic nutrient source; Proteomics

References