Nitrogen-to-Protein Conversion Factors for Crop Residues and Animal

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Nitrogen-to-Protein Conversion Factors for Crop Residues and Animal Manure Common in China Xueli Chen, Guanglu Zhao, Yang Zhang, Lujia Han, and Weihua XIAO J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03441 • Publication Date (Web): 02 Oct 2017 Downloaded from http://pubs.acs.org on October 3, 2017

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Journal of Agricultural and Food Chemistry

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Nitrogen-to-Protein Conversion Factors for Crop Residues and Animal Manure

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Common in China

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Xueli Chen, Guanglu Zhao, Yang Zhang, Lujia Han, and Weihua Xiao*

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Biomass and Bioresource Utilization Laboratory, College of Engineering, China

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Agricultural University, P.R. China 100083

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Corresponding Author * E-mail: [email protected]; Phone: +86 10 6273 6778; Fax: +86-10-6273-6778

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ACS Paragon Plus Environment


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ABSTRACT: Accurately determining protein content is essential in exploiting

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biomass as feed and fuel. A survey of biomass samples in China indicated protein

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contents from 2.65 to 3.98 % for crop residues and from 6.07 to 10.24 % for animal

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manure of dry basis. Conversion factors based on amino acid nitrogen (kA) ranged from

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5.42 to 6.00 for the former and from 4.78 to 5.36 for the latter, indicating that the

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traditional factor of 6.25 is not suitable for biomass samples. On the other hand,

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conversion factors from Kjeldahl nitrogen (kP) ranged from 3.97 to 4.57 and from 2.76

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to 4.31 for crop residues and animal manure, respectively. Of note, conversion factors

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were strongly affected by amino acid composition and levels of nonprotein nitrogen.

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Thus, kP values of 4.23 for crop residues, 4.11 for livestock manure, and 3.11 for

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poultry manure are recommended to better estimate protein content from total nitrogen.

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KEYWORDS: crop residues, animal manure, amide acid, protein, nitrogen-to-protein

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conversion factor

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1. INTRODUCTION

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Crop residues and animal manure, which are widely and abundantly available,1,2

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have both actual and potential value as source of proteins. For example, crop residues

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are frequently used as animal feed and nitrogen source for anaerobic fermentation

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because of their high protein content.3 Animal manure, similarly rich in protein, is also

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well known as a significant source of bioenergy, as fertilizer, or as animal feed.4-6

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Indeed, many forms of biomass are potentially useful as such, but have unknown

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protein content. Therefore, it is essential to develop a rapid and reasonably accurate

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method to determine protein content. Presently, there are four main analytical methods

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for food labeling purpose,7 including copper- or dye-based spectroscopy, UV- or

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IR-based techniques, amino acid analysis, and conversion of nitrogen content to protein

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content.

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Total nitrogen analysis by the Kjeldahl method is still widely favored as a basis of

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protein content determination, but its accuracy depends on nitrogen-to-protein

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conversion factors. In previous articles, Fujihara and colleagues8-10 reported

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nitrogen-to-protein conversion factors of 3.99 for mushrooms, 4.39 for vegetables, 5.75

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for rice, 5.81 for wheat, and 5.95 for other cereals. On the other hand, Diniz et al.11

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calculated conversion factors of 5.39 to 5.98 for nine species of fish from the Brazilian

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coast. There have also been many other reports12-14 demonstrating that the traditional

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conversion factor (kP) of 6.25 based on the assumption that the nitrogen content of

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proteins to be 16% is not suitable for estimating protein content in actual samples. In

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addition, most reported conversion factors were inferred from food and feed

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products,8-10,15-18 but rarely from biomass samples.12,19 Indeed, we know of very few

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conversion factors for crop residues or animal manure.

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Thus, in this study, we determined the amino acid composition, protein content, and

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three types of nitrogen-to-protein conversion factors (kA, kP, and k) for five crop

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residues and five types of animal manure. As defined by Mossé et al.,17 kA is the ratio of

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total anhydrous amino acids to total amino acid nitrogen, kP is the ratio of total

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anhydrous amino acids to total nitrogen, and k is the average of kA and kP. On the basis

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of the differences in these values, we now recommend new k factors more suitable for

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estimating protein content in crop residues and animal manure.

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2. MATERIALS AND METHODS

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2.1. Materials. A total of 50 representative crop residues, consisting of 10 samples

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each of wheat straw, rice straw, corn stover, rape stalk, and cotton stalk, were collected

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from different sites in China in 2011-2014. Each sample was collected at the fully ripe

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stage, with the grain and root removed and only the middle part retained, and

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thoroughly mixed to obtain a representative batch of approximately 2 kg. Subsequently,

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samples were dried for 36-48 h at 45 °C in a forced-air drying oven, per American

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Society for Testing and Materials E1757-01. Samples were then milled in a ZM100 mill

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fitted with a 0.90-mm sieve (Retsch GmbH & Company, Germany), and stored in

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plastic bags until analysis.20

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Fifty specimens of animal manure were also obtained from a variety of sources,21

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and consisted of 10 samples each of pig, dairy, beef, layer, and broiler manure. Each

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sample was collected from different sites on the unit floor in each barn, and a

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representative batch of about 2 kg was obtained after thorough mixing. The samples

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were then dried in a forced-air drying oven at 70 ± 5 °C for 18-24 h until there was no 4


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significant loss of moisture. After cooling, samples were directly ground in a ZM100

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mill (Retsch GmbH & Company, Germany), and passed through a 0.5-mm sieve.

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Finally, samples were stored in tightly sealed containers until analysis.21

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2.2. Analysis. Total nitrogen was determined according to the Official Methods of

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Analysis of AOAC International,22 using a Kjeltec 2300 auto-analyzer (FOSS Tecator

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AB, Sweden). To quantify amino acids, samples were hydrolyzed in 6 N HCl at 120 °C

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for 22 h, and analyzed by high performance liquid chromatography at 40 °C on an

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Agilent 1100 (USA) chromatography system fitted with a quaternary pump, an

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autosampler, a thermostatted column compartment, an online vacuum degasser, an

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ultraviolet (UV) detector, and a reversed-phase column (Agilent Hypersil ODS, 250

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mm × 4.0 mm × 5 µm). Tryptophan was determined by hydrolysis in Ba(OH)2 and

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subsequent acidification with HCl.23 Asparagine and glutamine levels were measured

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by assigning moles of amide nitrogen on a proportional basis to the moles of aspartic

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acid and glutamic acid present.18 Proline was quantified at 262 nm on HPLC, while all

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others

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acetate:trimethylamine:tetrahydrofuran (500:0.11:2.5, v:v:v) as solvent A and 80.9 mM

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sodium acetate:methanol:acetonitrile (1:2:2, v:v:v) as solvent B. Both solvents were

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adjusted to pH 7.20 with acetic acid. Samples (10 µL) were eluted at 1.0 mL/min over a

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gradient of 8-50 % B until 17 min, 50-100 % B until 20.1 min, and 0 % B until 24.0

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min. To measure amide nitrogen, samples were hydrolyzed in 3 N HCl at 115 °C for 2

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h, and titrated for liberated ammonia, as described for total nitrogen.24

were

quantified

at

338

nm,

using

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27.6

mM

sodium


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2.3. Data Processing and Analysis. Box charts in OriginPro 8.0 were used to

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analyze amino acid composition and calculate nitrogen-to-protein conversion factors.

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SPSS 17.0 was used for one-way analysis of variance.

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3. RESULTS AND DISCUSSION

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3.1. Amino Acid Composition. The amino acid composition of five crop residues

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and five types of animal manure is presented in Figure 1 as mean of 10 samples each.

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Glutamic acid (Glu) was the most abundant in all crop residues except cotton stalk, in

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which aspartic acid (Asp) was the most abundant instead (Figure 1A and Table S1).

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Similar trends were described by Lourenco et al. for tropical seaweeds.25 In rice straw

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and corn stover, Glu represented 0.65 % and 0.64 % of the dry basis, respectively, in

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line with data obtained for rice and corn, in which the amino acid represents 0.41-0.99 %

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and 0.38-1.57 % of the dry basis, respectively.10,18 In wheat straw, Glu accounted for

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0.55 %, a level lower than that typically found in wheat (1.00-1.97 %). Levels of

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cysteine (Cys), methionine (Met), asparagine (Asn), and glutamine (Gln) were notably

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low in all residues, and lower than in cereal products, in which these amino acids

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comprised 0.26 %, 0.32 %, 0.39 %, and 1.70 % of the dry basis, respectively.10 Indeed,

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multiple reports indicate that cereal10 and common food items18 are rich in Asn and

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Gln, however, great variety was found in different species. According to the literature,

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wheat had high Gln level but low Asn content,10 while banana showed less both Gln

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and Asn,18 which was in line with the low Gln and Asn contents of our data. As 6


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reported by Fujihara et al.,10 for rice, Glu was the predominant amino acid in rice straw,

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although Gln was much less abundant in the latter.

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Similar trends were observed in animal manure (Figure 1B and Table S2), except that

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average alanine (Ala) content was lower. Glu, Arg, Leu, Asp, and Pro constituted over

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half of total amino acids in dairy and beef manure. On the other hand, pig manure, layer

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manure, and broiler manure, in which Asp and Glu were the major amino acids,

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contained higher levels of Arg, Asp, and Gly compared to other manure types.

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Interestingly, layer manure was particularly rich in Arg, which constituted more than

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24 % of total amino acids. Finally, the concentration of most amino acids was relatively

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higher in pig and broiler manure than in other manure types.

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On average, Glu, Asp, Arg, and Leu constituted over 40 % of total amino acids in

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five crop residues and five types of animal manure, as was observed in algal biomass

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tested by Templeton and Laurens.19

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3.2. Protein Content. Protein content, listed in Table 1 as total anhydrous amino

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acids, ranged from 2.65 % of the dry basis in rape stalk to 3.98 % in cotton stalk. Of

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note, these values are lower than those typical of cereal products,10,15,18,26 but consistent

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with those of leaves in 90 plant species12 (Table 2). On the other hand, protein content

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was strongly variable in animal manure, with mean values ranging from 6.07 % in dairy

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manure to 10.24 % in broiler manure. Pairwise comparison showed that broiler manure

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and layer manure were significantly richer in protein (10.24 ± 2.90 % and 9.84 ±

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1.79 %, respectively) than dairy manure (6.07 ± 3.06 %). These differences are 7



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probably due to multiple factors, including growth stage, animal diet, and housing27,28.

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In any case, crop residues rich in protein are generally considered nutritionally useful as

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animal feed. Indeed, corn stover is frequently used as feed.20 Moreover, proteins in both

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crop residues and animal manure are essential to produce large amounts of ammonia

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nitrogen and to stabilize the pH during anaerobic fermentation.3

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The protein nitrogen fraction, calculated as amino acid nitrogen/total nitrogen

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(Figure 2), averaged 74.22 % for crop residues and 71.80 % for animal manure, in line

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with those of vegetables,9 algae,19 and plant leaves,12 although rape stalk (84 %) and

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broiler manure (54 %) had exceptionally high and low protein nitrogen fractions,

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respectively. However, protein nitrogen fractions were extremely low compared to

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those of food (89 %)18 and cereal products (93 %).10

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3.3. Nitrogen-to-Protein Conversion Factors. To determine protein content

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conveniently and accurately, the following nitrogen-to-protein conversion factors were

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calculated: kA, the ratio of protein to amino acid nitrogen,24 kP, the ratio of protein to

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total nitrogen,16 and k, the average of kA and kP.17 Ideally, these conversion factors

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should be independent of the protein or nitrogen content of biomass (Table 1).

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3.3.1. kA. The mean value of kA ranged from 5.42 to 6.00, with average 5.72 ± 0.39

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for five crop residues, in close agreement with the general conversion factor of 5.7 for

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wheat,30 animal, and plant products.18 Although significant differences among crop

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residues were not observed, kA was lower for crape stalk (5.42 ± 0.24) than for rice

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straw (6.00 ± 0.35). kA was also generally lower in animal manure, ranging from 4.78 8


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for layer manure to 5.36 for beef manure. On average, the kA value for animal manure

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(5.16 ± 0.31) was similar to that for green algae (5.13 ± 0.39)23. Values were

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comparable for dairy (5.33 ± 0.11) and beef manure (5.36 ± 0.09), but was lower for

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layer manure (4.78 ± 0.43). Such differences in kA are likely due to differences in

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amino acid composition.19 Strikingly, the kA we obtained for crop residues and animal

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manure was far from the traditional factor of 6.25. Furthermore, we note that kA can not

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be used to estimate the true protein content from total nitrogen, because it does not

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consider nonprotein nitrogen.9,10

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3.3.2. kP. The conversion factor kP is more useful in practice, since it enables direct

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estimation of protein content from total Kjeldahl or combustion (Dumas) nitrogen. For

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the five crop residues we tested, we obtained mean values ranging from 3.97 to 4.57,

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with overall average 4.23 ± 1.06. Significant variability was also observed among

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manure types, with average values ranging from 2.76 for broiler manure to 4.31 for

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dairy manure and significantly lower in comparison to those for crop residues.

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Importantly, the kP values we obtained were significantly lower than the traditional

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conversion factor of 6.25 due to the presence of nonprotein nitrogen. Instead, these

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values are similar to those calculated for microalgae (2.53-5.77),31-33 tropical plants

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(3.7-5.0),34 and various vegetables and mushrooms (2.38-5.84).8,9 Higher values

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between 4.74 and 6.26 have also been reported for cereal products, meat, fish, egg, and

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edible insects.10,18,35

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3.3.3. k. A mean factor (k) of the two main usual factors (kA and kP) has been

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proposed as a good compromise because kA and kP are higher and lower than the true

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factor.17 To improve reliability and comparisons of conversion factors among different

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species, the conversion factor k was also calculated here. The k values for wheat straw

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(4.94), corn stover (4.95), rape stalk (4.99), and cotton stalk (4.91) were similar to those

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reported for wheat germ (4.99)36 and wheat bran (4.96)13. Mossé17 reported a k value of

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5.1 for rice grains, which is close to that we obtained for rice straw (5.19). Interestingly,

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mean k values were lower for animal manure (3.92-4.82) than for crop residues

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(4.91-5.19), although variability was higher among the former.

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3.3.4. Choice of Conversion Factor. The choice of conversion factor may

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significantly impact the estimate of protein content in any sample (Figure 3). For

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example, kA should be considered for purified proteins, which contain very low levels

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of nonprotein nitrogen. On the other hand, kP may be more suitable for low-protein

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sources that are also rich in nonprotein nitrogen,13,15,17 such as the biomass samples we

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tested (Figure 2). However, kP values were significantly different (p < 0.05) between

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livestock (pig, dairy, and beef) and poultry manure (layer and broiler) and also highly

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variable (Figure 3D), with average values 4.11 ± 0.68 and 3.11 ± 0.87, respectively.

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Therefore, consensus kP values of 4.11 and 3.11 are recommended for livestock and

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poultry manure. Variability was also observed in kP values for five crop residues, but

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not to a statistically significant extent (p > 0.05), with average 4.23 ± 1.06 (Figure 3C).

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Accordingly, a kP of 4.23 may provide a reliable estimate of protein content in crop

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residues.

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3.4. Summary. The average protein content of five crop residues ranged from 2.65 %

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to 3.98 %, in agreement with published data for plant leaves, but was lower than that

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for cereal products. A large variation in protein content was observed in animal manure,

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with values ranging from 6.07 % to 10.24 %. Nitrogen-to-protein conversion factors

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based on amino acid nitrogen (kA) varied from 5.42 to 6.00 for crop residues and from

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4.78 to 5.36 for animal manure, indicating that the general conversion factor of 6.25 is

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not suitable for biomass samples. On the other hand, the more practical conversion

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factor based on the proportion of total anhydrous amino acids to total nitrogen (kP)

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ranged from 3.97 to 4.57 and from 2.76 to 4.31 for crop residues and animal manure,

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respectively, and are much lower than the traditional factor of 6.25 due to the presence

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of nonprotein nitrogen. Hence, kP values of 4.23 for crop residues, 4.11 for livestock

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manure, and 3.11 for poultry manure are recommended to reliably estimate biomass

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protein from total nitrogen. These conversion factors should provide a quick and

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reasonably accurate method of estimating the protein content in crop residues and

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animal manure, and should facilitate their efficient use as feed or fuel.

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ASSOCIATED CONTENT

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Supporting Information

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Table S1. Amino Acid Composition and Total Amino Acid Content (g/100 g Dry

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Basis) of Crop Residues

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Table S2. Amino Acid Composition and Total Amino Acid Content (g/100 g Dry

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Basis) of Animal Manure

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AUTHOR INFORMATION

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Funding

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We acknowledge the National Key R&D Program of China (No. 2016YFE0112800), the

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European Union’s Horizon 2020 Research and Innovation Programme (No. 690142),

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Project AgroCycle (Sustainable Techno-Economic Solutions for the Agricultural Value

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Chain), and National Natural Science Foundation of China (No. 31671572).

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Notes

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The authors declare no competing financial interest.

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ABBREVIATIONS USED

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kA, ratio of anhydrous amino acids to amino acid nitrogen; kP, ratio of anhydrous amino

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acids to total nitrogen; k, average of kA and kP.

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Sijtsma, L.; Cifuentes, A.; Herrero, M.; Ibañez, E. Downstream processing of

324

Isochrysis galbana: a step towards microalgal biorefinery. Green Chem. 2015, 17,

325

4599-4609.

326 327

(34) Milton, K.; Dintzis, F. R. Nitrogen-to-protein conversion factors for tropical plant samples. Biotropica 1981, 13, 177-181.

328

(35) Janssen, R. H.; Vincken, J. P.; van den Broek, L. A.; Fogliano, V.; Lakemond,

329

C. M. Nitrogen-to-protein conversion factors for three edible insects: Tenebrio molitor,

330

Alphitobius diaperinus, and Hermetia illucens. J. Agric. Food Chem. 2017, 65,

331

2275-2278.

17



332 333

(36) Tkachuk, R. Nitrogen-to-protein conversion factors for cereals and oil-seed meals. Cereal Chem. 1969, 46, 419-423.

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Page 18 of 25

Page 19 of 25


334

FIGURE CAPTIONS

335

Figure 1. The averaged value of each amino acid for (A) five crop residues; (B) five

336

types of animal manure. All data are shown as % contribution to weight of dry basis.

337

Figure 2. Protein nitrogen fraction in biomass tested in this and previous studies (*

338

mushroom,8 vegetables,9 algal samples,19 plant leaves,12 food products,18 and cereal

339

products10).

340

Figure 3. Variability in kA (A,B), kP (C,D) and k (E,F) for crop residues (A,C,E) and

341

animal manure (B,D,F). Middle lines and hollow squares within the box mark the

342

median and mean, respectively, and the lower and upper boundaries represent the 25th

343

and 75th percentile. Error bars above and below the box indicate 90 and 10 percentiles

344

of all data, while diamonds inside and outside the box are > 90 and < 10 percentiles.

345

19



Page 20 of 25

TABLES

Table 1. Protein Content and Nitrogen-to-Protein Conversion Factor for Crop Residues and Animal Manurea Proteinb

nitrogen recovery

nitrogen-to-protein conversion factorsc

(wt% of dry basis)

samples

sample

(wt% of dry size

Kjeldahl basis)

amino acids

kA

kP

k

0.76 ± 0.44

5.78 ± 0.43

4.10 ± 0.59

4.94 ± 0.40

10

nitrogen crop residues wheat straw

3.12 ± 1.84

0.53 ± 0.28

rice straw

3.84 ± 0.95

0.65 ± 0.19

0.89 ± 0.22

6.00 ± 0.35

4.37 ± 0.59

5.19 ± 0.26

10

corn stover

3.50 ± 0.81

0.61 ± 0.15

0.89 ± 0.23

5.72 ± 0.38

3.97 ± 0.54

4.95 ± 0.32

10

rape stalk

2.65 ± 1.02

0.49 ± 0.19

0.62 ± 0.29

5.42 ± 0.24

4.57 ± 1.19

4.99 ± 0.69

10

cotton stalk

3.98 ± 1.63

0.71 ± 0.29

1.00 ± 0.20

5.68 ± 0.37

4.13 ± 1.88

4.91 ± 0.91

10

total sample

3.42 ± 1.35

0.60 ± 0.23

0.83 ± 0.31

5.72 ± 0.39

4.23 ± 1.06

4.97 ± 0.56

50

pig manure

9.19 ± 3.25

1.74 ± 0.61

2.27 ± 0.75

5.28 ± 0.07

4.04 ± 0.43

4.66 ± 0.23

10

dairy manure

6.07 ± 3.06

1.13 ± 0.55

1.47 ± 0.74

5.33 ± 0.11

4.31 ± 0.97

4.82 ± 0.51

10

beef manure

7.72 ± 1.77

1.44 ± 0.33

1.94 ± 0.37

5.36 ± 0.09

3.99 ± 0.56

4.67 ± 0.29

10

layer manure

9.84 ± 1.79

2.09 ± 0.48

2.94 ± 0.68

4.78 ± 0.43

3.45 ± 0.72

4.11 ± 0.38

10

broiler manure

10.24 ± 2.90

2.03 ± 0.60

4.10 ± 1.44

5.08 ± 0.20

2.76 ± 0.90

3.92 ± 0.50

10

total sample

8.61 ± 2.96

1.69 ± 0.62

2.54 ± 1.24

5.16 ± 0.31

3.71 ±0.90

4.44 ± 0.52

50

animal manure

a

b

The results are recorded as the mean ± standard deviation. Sum of anhydrous amino acids, and represents the true protein

content. ckA is the ratio of protein to amino acid nitrogen; kP is the ratio of protein to total Kjeldahl nitrogen; k is the average of kA and kP.

20


Page 21 of 25


Table 2. Reported Protein Content of Common Cereal Products and Plant Leaves samples

protein content (wt% of dry basis)

reference

wheat

8.46 – 14.10

10,15,18

rice

4.82 – 8.06

10,18

corn

5.30 – 8.61

10,18,26

plant leaves

0.20 – 7.45

12,29

21



Page 22 of 25

FIGURES

% weight of dry basis

A

0.7

Wheat straw Rice straw Corn stover Rape stalk Cotton stalk

0.6 0.5 0.4 0.3 0.2 0.1 0.0

Ala Arg Asp Cys Glu Gly His

Ile

Leu Lys Met Phe Pro Ser Thr Trp Tyr Val Asn Gln

% weight of dry basis

B 3.0 Pig manure Dairy manure Beef manure Layer manure Broiler manure

2.5 2.0 1.5 1.0 0.5 0.0

Ala Arg Asp Cys Glu Gly His

Ile

Leu Lys Met Phe Pro Ser Thr Trp Tyr Val Asn Gln

Figure 1

22


Page 23 of 25


Protein nitrogen fraction

Nonprotein nitrogen fraction

100% 80% 60% 40% 20% 0%

Figure 2

23



Figure 3

24


Page 24 of 25

Page 25 of 25


TOC GRAPHIC

25


Nitrogen-to-Protein Conversion Factors for Crop Residues and Animal

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