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We have validated an O-phthalaldehyde (OPA)-based derivatization method, showing accurate and linear quantification for standard reference amino acids...
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New Analytical Methods

Total Protein Analysis in Algae via Bulk Amino Acid Detection: Optimization of Amino Acid Derivatization after Hydrolysis with O-Phthalaldehyde 3-Mercaptopropionic Acid (OPA-3MPA) Hunter Cuchiaro, and Lieve M. L. Laurens J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00884 • Publication Date (Web): 24 Apr 2019 Downloaded from http://pubs.acs.org on May 5, 2019

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Journal of Agricultural and Food Chemistry

Total Protein Analysis in Algae via Bulk Amino Acid Detection: Optimization of Amino Acid Derivatization after Hydrolysis with O-Phthalaldehyde 3-Mercaptopropionic Acid (OPA3MPA)

Hunter Cuchiaro§, and Lieve M.L. Laurens* National Renewable Energy Laboratory, 15013 Denver West Parkway, Golden, Colorado, USA * Author for correspondence; Phone: +1 (303) 384-6196; email: [email protected] § current

address: 1301 Center Ave, Colorado State University, Fort Collins, CO 80523

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Abstract

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The analysis of protein in algal biomass is one of the most critical areas of commercial

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development of algae characterization for nutritional or other high value applications. A new

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rapid and accurate method is required that can be widely implemented and that is free of

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interferences from the complex algal biomass matrix. We developed a simple spectrophotometric

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method for primary amino acid quantification bulk measurement in an acid hydrolyzed algal

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biomass preparation, as an alternative to the more labor-intensive amino HPLC acid analysis or

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less specific nitrogen-to-protein conversion. We have validated an O-phthalaldehyde (OPA)-

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based derivatization method, showing accurate and linear quantification for standard reference

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amino acids as well as mixtures, mimicking the amino acid complexity found in algal biomass.

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The presence of interferences that may be derived from the complex biomass biochemical

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composition was tested during the method validation phase. We document the application of a

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novel method of OPA derivatization with 3-mercaptopropionic acid (MPA) to determine the total

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amino acid content of harvested algal biomass collected from different, controlled cultivation

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conditions and demonstrated a within 10% accuracy against a reference measurement of amino

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acid content in at least 4 species and 10 algal biomass samples, across early, mid and late-stage

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of cultivation.

18 19

Acronyms

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IEC – Ion Exchange Chromatography; RP-HPLC – Reverse Phase High Performance Liquid

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Chromatography; OPA – O-phthalaldehyde; 2ME – 2-mercaptoethanol; 3MPA – 3-

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mercaptopropionic acid; AA – amino acid; NPN – Non-protein nitrogen.

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Keywords

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Amino Acid Analysis; Algae; OPA; o-phthalaldehyde; Spectrophotometry.

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Background The protein content of algae has a strong influence on determining the economic potential

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for food and feed uses for algal biomass and thus accurately determining protein content is

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critically important. The protein content of microalgae can range from 7% to 40% and can

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change dramatically over the course of its lifecycle, however, the biochemical composition of the

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biomass is highly complex and thus many analytical methods suffer significant interferences

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when characterizing different individual constituents.1–6 For example, it is known that there are

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significant interferences from reducing substances, such as sugars, on the color-development of

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the bicinchoninic acid (BCA) colorimetric protein method based on Lowry’s procedure.7,8 The

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presence of reducing sugars in the hydrolyzed whole biomass used for protein determination is

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thought to be responsible for an up to two-fold overestimation of protein content when compared

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against total amino acid content of the same biomass.8 Those spectrophotometric methods

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referred to as the Lowry et al. or Bradford are also of limited utility unless used to quantify

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purified protein, as measured by dry weight estimation.7,9,10

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Total protein content can be determined via a nitrogen-to-protein conversion factor,

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which is typically derived from the respective amino acid composition of algal biomass.11 Amino

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acid analysis is a common analysis when characterizing biomass and is usually done by the use

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of ion exchange chromatography (IEC) or reverse phase liquid chromatography (RP-HPLC)

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followed by a post-column ninhydrin derivatization. Ninhydrin reacts with the sample and the

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mixture is heated to 100 ºC, and the resulting Ruhemann’s purple product is thereby measured

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colorimetrically.12–16 This established method can be applied to separations or bulk materials, but

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high oxygen and light sensitivity, slow reaction time at high temperature, and high background

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absorbance are disadvantageous, as is the need for specialized and dedicated instrument set up.17

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Ninhydrin is also known to react with imines, guanidino groups (found in arginine), amides

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(found in asparagine and glutamine), indole rings (tryptophan), sulfhydryl groups (cysteine),

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cytosine, guanine, and cyanide ions to form chromophores and thus linking ninhydrin

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derivatization detection with HPLC separation is appropriate to reduce these interferences.16

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Existing methodologies based on combustion (e.g. Dumas method) are widely used to

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determine protein but often rely on a dedicated nitrogen conversion factors, based on the

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nitrogen content of each amino acid.11,18 Amino acid analysis can be complex, labor intensive

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and is sometimes out of reach for rapid characterization of biomass to laboratories that are not

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equipped with complex instrumentation, such as the HPLC instrumentation to support the

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analysis of individual amino acids. Amino acid derivatization itself can be performed easily at

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mild temperatures with appropriate derivatization agents and as long as no other products that are

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present interfere with the colorimetric reaction.

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In the early 1970’s a method based on O-phthalaldehyde (OPA) and 2-mercaptoethanol

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(2ME) was developed as an alternative method for amino acid (AA) detection. The fluorescent

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adduct was specific to primary amines.19,20 The greater specificity of the OPA/thiol/AA adduct is

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advantageous over ninhydrin when considering complex samples, by simplifying sample

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preparation.21 Additionally, proline, with its unique pyrrolidinyl structure, can be measured after

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perchloric acid pre-treatment.22,23 The primary disadvantage of this method was that the analyte

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is an intermediate that decays to a non-productive final product.24 Much work has been done to

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study the kinetics of this reaction, and it is often stated in the literature that measurements should

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be performed at the same timepoint to preserve accuracy.25–27 The adduct is stabilized by

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substituting Roth’s original 2ME for 3-mercaptopropionic acid (3MPA), reaction mechanism is

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shown in Figure 1.28

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A review of the literature revealed that no study had been carried out using OPA/3MPA

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derivative absorbance to quantify total amino acids in a bulk preparation without the

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implementation of a liquid chromatography.29–34 The objective of this work was to develop and

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optimize a simple spectrophotometric method for the bulk determination of amino acids in algal

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biomass as an approximation of total protein content. UV/Vis spectrophotometry is a widely

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available, low-cost technique that is well suited to high-throughput analysis. The absorbance of

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OPA/AA derivatives could be quantified using a summation of relative absorbance response

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factors.35 When comparing amino acid profiles across 14 species and growth conditions,

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previous research in our group found that the normalized weight percentage of amino acids was

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relatively stable.11 Arginine, lysine, and alanine had the greatest range in normalized weight

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percentage.11 It was hypothesized that variations in individual amino acid content between

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species and growth condition would not substantially affect the overall absorptivity of analytical

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mixtures and thus a bulk measurement based on amino acid derivatization may prove a

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promising approach.

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The objectives of this work were to optimize a method for OPA/AA derivatization,

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accurately measure amino acid content in bulk amino acids standards, investigate the impact of

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interfering compounds within process streams, and to compare this new method’s data accuracy

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on hydrolyzed protein from biomass against total amino acid data obtained from a commercial

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analytical laboratory.

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Materials and Methods

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Materials

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O-phthalaldehyde (P0657), 3-mercaptopropionic acid (M5801), L-arginine (A5006), L-

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aspartic acid (A9256), L-alanine (A7627), L-cysteine (C7352), L-glutamic acid (G1251), glycine

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(G8898), L-histidine (H8000), L-isoleucine (I2752), L-leucine (L8000), L-lysine (L5501), L-

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methionine (M9625), L-phenylalanine (P2126), L-serine (S4500), L-threonine (T8625), L-

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tryptophan (T0254), L-tyrosine (T3754), L-valine (V0500) were purchased from Sigma Aldrich.

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For all solution, nanopure water was used (18 MΩ deionized water). Methanol (A412P-4),

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sodium chloride (S271-500), ethanol (A4094), boric acid (A73-1), sodium nitrate (S343-500),

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ammonium nitrate (A676-500), sodium hydroxide (SS256-500), guanidinium hydrochloride

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(AAJ7582322) were purchased from Fisher Scientific. All reagents were ACS-grade or better.

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Absorbance measurements were taken using a Beckman Coulter DV 800

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spectrophotometer, equipped with a six-position automated cell holder, and 10mm pathlength,

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1.0 mL matched quartz cuvettes (50-823-023) purchased from Fisher Scientific. Single-

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wavelength absorbance measurements were made at 334 nm and absorbance spectra recorded

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between 600-250 nm.

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Solutions Preparation Diluent (20% 0.2M Sodium tetraborate, pH 9.3 / 80% Methanol) was prepared by dissolving 6 g boric acid in 480 mL water, and pH was then adjusted to 9.3 +/-0.5 with 5 M

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NaOH. 400 mL of this solution was combined with 1600 mL methanol and stirred until the

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solution returned to room temperature. The prepared solution was stored at room temperature in

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a flammables cabinet.

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OPA/3MPA Reagent Preparation OPA was prepared to a final concentration of 8mM in water and sonicated to dissolve. To

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the same flask, 3MPA was added so that the final concentration would be 24 mM. OPA/3MPA

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reagent was prepared fresh daily.

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Individual Amino Acid Stock Stock solutions of each amino acid (L-Aspartic Acid, L-Glutamic Acid, L-Threonine,

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L-Serine, L-Glycine, L-Alanine, L-Valine, L-Methionine, L-Leucine, L-Isoleucine, L-

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Tyrosine, L-Phenylalanine, L-Tryptophan, L-Lysine, L-Histidine, L-Arginine, L-Cysteine)

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were prepared at a concentration of 2mM in diluent using volumetric glassware. Solutions were

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stored at 5 ºC and used within two weeks of preparation.

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Bulk Amino Acid Stock Preparation

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When preparing bulk stock solutions, amino acids were divided into groups of four (A:

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L-Aspartic Acid, L-Threonine, L-Serine, L-Glutamate; B: L-Glycine, L-Alanine, L-Valine, L-

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Methionine; C: L-Isoleucine, L-Leucine, L-Tyrosine, L-Phenylalanine; D: L-Tryptophan, L-

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Lysine, L-Histidine, L-Arginine). Each bulk had concentration of 500 µM per amino acid, and

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the total concentration of each bulk solution was 2mM total amino acids using volumetric

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glassware. Stocks were sonicated for 10 minutes and vortexed for 30 seconds, repeated once.

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These are the intermediate stocks.

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Bulk analytical stock solutions were prepared by combining intermediate stocks A, B, C,

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and D 1:2:3:4 (stock 1), 2:3:4:1 (stock 2), 3:4:1:2 (stock 3), and 4:1:2:3 (stock 4). Replicates of

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each analytical stock were prepared together and stored at 5 °C.

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Interference Stocks Chloride, sulfate, nitrate, ammonium, hexose sugars (1:2 mannose:glucose), ethanol and

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guanidinium stocks were prepared at a concentration of 1.25 mM. Chloride, sulfate, nitrate,

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ammonium, and guanidinium were dissolved in deionized water. Hexose sugars and ethanol

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were dissolved in diluent.

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Sample Preparation Algal samples were prepared and analyzed in triplicate for 3 species (S. obliquus, N.

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oculata, and C. vulgaris) at early-, mid-, and late-stage growth, and D. aramatus at mid-stage

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growth. Replicates were prepared by weighing between 10 and 15 mg (± 1.5 mg) lyophilized

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algal biomass into Agilent borosilicate HPLC vials. For hydrolysis, 1 mL 6 N HCl was added to

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each sample. Vials were capped with PTFE septa and vortexed for 20 seconds. Vials were

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hydrolyzed at 110 ºC in a heating block for 24 hours, then vortexed for 20 seconds, uncapped,

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and set to dry under nitrogen. 1 mL methanol was added to each vial and samples were again

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vortexed for 20 seconds, then set to dry again under nitrogen. Samples were then placed under

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vacuum at 40 °C to finish drying overnight.

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Vials were returned to room temperature on benchtop (about 10 minutes). 1000 µL

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diluent was added to each sample, then immediately recapped. Samples were reconstituted by

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vortexing vials for 90 seconds each, then for an additional 20 seconds immediately prior to

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filtration through 0.2 µm PTFE syringe filters into clean HPLC vials.

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A total of 690 µL of biomass filtrate was diluted to final volume 5.0 mL in falcon tubes.

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Tubes were sealed and then vortexed to mix. This is the analytical sample stock. BSA samples

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were prepared using the same method, but with a dilution of 140 µL to 5mL. These are the

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analytical stock solutions. Approximately 1.5 mL of analytical stock solution was tranferred to

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clean, fresh HPLC vials to prepare individual replicates, capped and stored at -20 ºC.

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Analytical Dilutions

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For the individual and bulk amino acid standards, the reaction mixture consisted of a total

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volume of 5 mL, including amino acids from the stock at concentrations between 20 and 100 µM

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diluted with diluent, and brought to final volume with 2.5 mL of the OPA/3MPA stock solution

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(final concentrations of 4 mM OPA and 12 mM 3MPA respectively). Samples were vortexed for

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20 seconds after the addition of OPA/3MPA, and reaction times were measured relative to the

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completion of this step. Measurements were corrected by subtracting the average OPA

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interference collected over the course of the study. Interference stocks were diluted to final

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concentrations between 5 and 125 µM in the reaction mixture, in the presence of consistently 60

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µM amino acid bulk solution (as described above). For each of the hydrolyzed biomass samples,

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a total of 0.25 mL of analytical stock was combined with 2.25 mL diluent and 2.5 mL

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OPA/3MPA stock solution as described above. Measurement spectra were normalized by using

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diluent, OPA, and sample blanks.

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Results and Discussion

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Derivatization reaction optimization

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The derivatization reaction was optimized by measuring the absorbance of individual

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amino acid standard solutions. A calibration curve was made by diluting stocks between 20 and

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100 µM and measuring absorbance spectra in 15-minute intervals after the OPA/3MPA addition

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step between 0-90 minutes. Instrument response was observed to increase from 0-30 minutes,

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and then slowly decay from 30-90 minutes. Figure 2 shows characteristic spectra after 30-

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minute derivatization time, with absorbance peak at 334 nm. This was the analytical wavelength

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used for subsequent measurements. We calculated the linearity of the calibration curve at each

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timepoint for each amino acid. We also attempted this experiment using a pH 7.2

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phosphate/methanol (50/50 % v/v) diluent and confirmed that the lower pH is unsuitable for

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OPA/3MPA derivatization.

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Based on this initial study, we selected a window between 30-60 minutes for further

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study. Fresh individual amino acid standards were prepared following the same procedure and

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measured at 5-minute intervals from 30-55 minutes. The same general trend of signal decay was

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observed over the course of each measurement, which we attributed to the conversion of the

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AA/OPA/3MPA derivative to the non-absorbing final product reported in the literature.22 We

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then prepared an OPA/3MPA blank and measured the absorbance at the same timepoints. The

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absorbance of the OPA/3MPA blank decreased at a comparable rate as that measured in the

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derivatized standards. When this blank absorbance was subtracted from the absorbance of the

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derivatized standards, the rate of signal change over time was approximately 0.0003 AU/min.

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We found that this trend was mostly not observed in AA solution when an OPA/3MPA

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blank measurement was subtracted. The calibration curve slope and absorbance values remained

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largely consistent. The average of all OPA blanks throughout this study was calculated and

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subtracted from individual AA calibration curve measurements to refine the measurement.

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Linear regression analysis was performed on the absorbances of each calibration curve at all

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timepoints. The regression slopes for each amino acid were tabulated, and the correlation

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coefficient (R2) at each timepoint averaged across 16 amino acids (Table 1). The correlation

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coefficient (R2) was deemed suitable across all time points, ranging from 0.997 at 30 minutes to

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0.996 at 55 minutes, with an average value of 0.997.

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To assess the sensitivity of our method, the molar extinction coefficients for each amino

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acid derivative were calculated at each timepoint and concentration and averaged as shown in

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Table 2. The results of this calculation were tabulated and compared to those reported by

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Svedas.24 The extinction coefficient for 15 of the 16 amino acids was calculated to be greater

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than those previously reported, indicating the present method has on average 29% higher

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sensitivity with a less than 10% relative standard deviation of the measurements, suggesting no

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significant outliers in responses recorded. Tyrosine, the lone exception, had an extinction

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coefficient approximately equal to the previously reported value.

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Bulk amino acid stocks were analyzed at the same theoretical concentration as the

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individual calibration curves. We combined the individual amino acid calibration slopes in a

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weighted average based on the fractional concentrations within each bulk solution. This

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calculation was performed at each timepoint and used to measure derivatized solutions’

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absorbance from 30-55 min. The diluent blank and average OPA blank interference was

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subtracted from measurements for each timepoint. We then calculated the AA concentration by

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dividing the measured response by the predicted slope at each timepoint (Table 2). Measured

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concentration was also calculated using the average normalized slope for comparison. The

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calculated concentrations were within 4.7% of theoretical for solutions 1-3, and within 5.7% of

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theoretical for solution 4. It was observed that using the average total predicted slope to calculate

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each concentration produced a more precise measured value. Thus, the average predicted slope,

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calculated across all time points, was used for subsequent measurements. Figure 3 shows results

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from bulk solutions 1 and 3, where the concentrations of amino acid groups were inverted

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between preparations. Linearity was preserved in both cases, demonstrating that mixtures of

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amino acids could still be comparably measured even with differing molar absorptivities between

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species. When we performed an ANOVA on the variance of error for each of the concentration

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levels, we found no significant difference in variance distribution between the different

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concentration levels and thus, with the underlying assumption of normal distribution of the

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uncertainty, we conclude that there is no discernable trend of error with concentration.

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It is worth noting that analysis with OPA derivatives is often done at a fixed time

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following the introduction of OPA/3MPA reagent to sample solution. The data generated in this

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study do not indicate a drastic change in the measured AA concentration after the subtraction of

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the OPA blank interference over the 25-minute period presently studied.

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The subtraction of an average OPA blank from the bulk measurement was justified by a

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conformational study, where the bulk AA mixture was diluted at concentrations 24-96 µM

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(inside the original curve) and an OPA blank was prepared with samples. The diluent and OPA

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blanks were subtracted from the raw measurements, collected in triplicate, concentration

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calculated based on the predicted slope. This procedure was repeated on three separate days. The

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measurement error was not more than 6.2%, and the average measured concentrations had an R2

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of 0.9995 across all three tests.

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As a theoretical exercise, we modeled the expected sensitivity for each of the 14 algal

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species/growth conditions previously reported at 30-55 minutes after derivatization.11 This was

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done by multiplying the experimentally-derived slopes of the 16 amino acids in this study by

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their reported concentration for in each species, and adding them to find the total. An average

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expected slope of 0.0070 µM/AU was calculated with an RSD of 0.7% for determining the

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amino acid concentration in algal biomass, based on these 14 species/growth conditions. This

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was the response factor used in amino acid content calculation of algal biomass samples.

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Limit of detection and quantification Further studies were performed using the bulk analytical standards to determine limits of

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quantitation and detection. Experimental results were linear and met AOAC suitability percent

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recovery criteria between 4.0 and 200 µM. The LOD and LOQ were also calculated based on the

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average OPA blank standard deviation at each timepoint. This method produced an average LOD

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of 2.1 µM and LOQ of 7.0 µM AA.

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Interference of components in process streams

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We assessed potential interferants that may be present during biomass processing.

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Depending on the process stream, potentially interfering species could be introduced as reagents

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or decomposition products. These studies included sulfate, chloride, ethanol, hexose sugars (1:2

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mannose:glucose), nitrate, guanidinium, and ammonium, all potentially present after algal

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biomass conversion.36 Only ammonium was predicted to interfere with AA measurement, based

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on Roth’s original work also demonstrated reactivity of NH4+ with OPA.19 We had suspected

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that lysine’s guanidinyl functional group had some activity with OPA, thus leading to the

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derivative’s high molar absorptivity, the experimental data indicated that only ammonium was

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observed to interact with the OPA (Figure 4).

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Algal biomass amino acid determination

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We tested a total of 10 algal species/growth conditions for amino acid determination in

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this study. Scenedesmus, Nannochloropsis, and Chlorella strains were each harvested at early,

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middle, and late growth stages, corresponding to high protein, carbohydrate, and lipid content in

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the biomass composition. The Desmodesmus armatus strain was harvested at a middle growth

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stage, corresponding to higher carbohydrate content. Freeze dried biomass samples were sent out

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to a contract analytical laboratory for quantitative total amino acid analysis using the ninhydrin

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reaction. We additionally included bovine serum albumin (BSA), a known and well-

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characterized and purified reference protein, as a control protein sample for hydrolysis, in

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addition to the algal biomass as a check standard and compared our results to the label claim of

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96-99% protein. The absorbance spectra of the derivatized hydrolysis products for each of the 4

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algae samples is presented in Figure 5.

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We performed optimization studies on the hydrolysis method to improve hydrolysis

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yield. We compared measured weight percent amino acid of Desmodesmus when hydrolyzed in 6

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N HCl (control), 0.5% (w/v) phenol/6 N HCl, and 0.5% (w/v) 3MPA/6N HCl for 24 hours, and

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also 6 N HCl for 18 hours.37 The 18 hour hydrolysis time produced a similar average

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measurement but was less precise than the 24 hour hydrolysis time. The range between analytical

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replicates was 25% greater for the 18 hour hydrolysis time of the same biomass. Additionally,

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we tried filtering the hydrolysate and rinsing with 0.5 mL methanol prior to drying down under

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nitrogen. None of these conditions produced a more optimal yield than the control, and in fact,

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filtering/rinsing the hydrolysate prior to drying down effectively decreased the measured amino

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acid yield by approximately 40%.

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It was expected that hydrolyzed biomass would produce a systematic over-measurement

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of protein content due to some interference from non-protein nitrogen. Ammonia and other

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primary amines evolved from the strong acid (HCl) digestion of asparagine, glutamine, histidine,

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tryptophan, chlorophylls, or nucleic acids could interfere with the accuracy of our method.

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During acid hydrolysis, glutamine and asparagine are converted to glutamic acid and aspartic

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acid respectively, and in the data obtained from our contract analytical laboratory both forms are

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reported as a single respective measurement and thus it is impossible to estimate the contribution

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of ammonium released from acid hydrolysis.

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It is difficult to quantifiably estimate the primary mechanism of non-protein nitrogen

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interference in this method. To account for any ammonium released by acid digestion of

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nitrogen-containing amino acids, which is not typically reported by a contract analytical

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laboratory, we applied a normalization factor based on the BSA control. Using this

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normalization factor, our method’s over-estimation compared to contract lab analysis was

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reduced to 11.2%. A BSA digestion was performed with each acid hydrolysis and applied to

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those samples respectively. The BSA normalization factor was not averaged across multiple

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analyses. Alternatively, we also considered a normalization scheme to account for ammonia

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released from acid digestion of non-protein nitrogen (NPN) sources. We averaged 21 NPN

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measurements previously reported in the literature and estimated an average level of 40.6% by

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weight NPN across multiple species/timepoints.11 Since some of the NPN would manifest as

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ammonia and some as secondary amines, nitrate or guanidine, we made the conservative

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estimate that half of the NPN would interfere with our measurement. This 20.3% NPN correction

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produces a more accurate measurement of the overall protein content compared to contract

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laboratory results (average 7.9% over-estimation) but does not account for ammonia released

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from protein decomposition. It is likely that ammoniac interference is the result of some

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combination of protein degradation products and NPN from the biomass. Further optimization

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studies may be warranted to determine the contribution specific to each factor (Figure 6).

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This measurement assumes total amino acid recovery from 6 N HCl hydrolysis, and the

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calibration curve was constructed based on 16 amino acids. These assumptions were made to

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model the biomass hydrolysate but may contribute to the overestimation of protein content in the

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sample.

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Our method utilizes a simple procedure for sample preparation and analysis of protein

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content in freeze-dried algal biomass with high sensitivity and precision, across multiple phyla

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and 10 species/growth conditions. Analyses of bulk mixtures of standards indicates high

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accuracy and linearity of our method from 8-200 µM amino acids. Corrections for non-protein

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nitrogen and in particular ammonia interference in the biomass hydrolysates is necessary,

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however this method is suitable for the rapid estimation of the bulk protein content in algal

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biomass. Both analytical and computational suggestions are made for further optimization of this

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analysis in future studies.

337 338

Acknowledgements

339

We would like to acknowledge Nick Sweeney and Stefanie Van Wychen for helpful discussions,

340

as well as the NREL Research Participant Graduate Internship program for making this project

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possible. This work was authored by the National Renewable Energy Laboratory and financially

342

supported by the U.S. Department of Energy under Contract No. DE-AC36-08GO28308 with the

343

National Renewable Energy Laboratory, as part of the DOE Office of Energy Efficiency and

344

Renewable Energy, Bioenergy Technologies Office. The views expressed in the article do not

345

necessarily represent the views of the DOE or the U.S. Government. The U.S. Government

346

retains and the publisher, by accepting the article for publication, acknowledges that the U.S.

347

Government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or

348

reproduce the published form of this work, or allow others to do so, for U.S. Government

349

purposes.

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Vasanits, A.; Kutlán, D.; Sass, P.; Molnár-Perl, I. Retention/Quantitation Properties of the o-Phthaldialdehyde-3-Mercaptopropionic Acid and the o-Phthaldialdehyde-N-Acetyl-LCysteine Amino Acid Derivatives in Reversed-Phase High-Performance Liquid Chromatography. J. Chromatogr. A 2000, 870 (1–2), 271–287.

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Figures Figure 1: Reaction mechanism forming the amino acid, O-phthalaldehyde/mercaptopropionic acid (AA/OPA/MPA) analyte

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Figure 2: Characteristic absorption spectrum of AA/OPA/MPA derivative. An absorbance peak was observed at 334nm and selected as the analytical wavelength for subsequent analyses in this study.

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Figure 3: (A) Absorbance versus concentration plots for bulks amino acid solutions # 1, 2, 3, and 4 (as described in the text). The R2 values for each were 0.9999, 0.9994, 0.9996, and 0.9999, respectively. (B) Error (%) of measured protein concentration versus theoretical calculated concentration.

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Figure 4: Matrix interference based on specific process products; OPA derivatization recovery was tested in the presence of 7 compounds typically present in algae-derived process streams, based on a reference sample at 60 µM amino acids

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Figure 5: Spectral interference analysis based on absorbance spectra of early stage harvest C. vulgaris (A), S. obliquus (B), N. oculata (C), and D. armatus (D), following acid hydrolysis and OPA/MPA derivatization

A

B

Chlorella vulgaris

2

Scenedesmus obliquus

2

Absorbance (AU)

Diluent OPA/MPA Matrix

1

1

Replicate 1 Replicate 2 Replicate 3

0

0 250

C

300 350 Wavelength (nm)

D

Nannochloropsis oculata

1

300 350 Wavelength (nm)

400

Desmodesmus armatus

2

Absorbance (AU)

2

250

400

Diluent OPA/MPA Matrix Replicate 1 Replicate 2 Replicate 3

1

0 250

300 350 Wavelength (nm)

400

0 250

300 350 Wavelength (nm)

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Figure 6: Protein measurements of algal biomass following 6N acid digestion, compared to contract laboratory. Species/timepoints are as follows: 1-Chlorella vulgaris (early); 2- Chlorella vulgaris (mid); 3- Chlorella vulgaris (late); 4- Scenedesmus obliquus (early);5- Scenedesmus obliquus (mid); 6-Scenedesmus obliquus (late); 7- Nannochloropsis oculata (early); 8Nannochloropsis oculata (mid);9- Nannochloropsis oculata (late); Desmodesmus armatus (mid), data shown as mean ± stdev of triplicate analyses for all samples

50.0

Weight % Amino Acids

40.0

30.0 Contract Lab Reported Measured AA Content BSA Normalization

20.0

20.3% NPN Normalization

10.0

0.0 1

2

3

4

5

6

7

8

9

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Tables Table 1: Typical blank-corrected absorbance data for AA/OPA/MPA derivative (Glutamate shown), between 30-55 minutes for 4 different amino acid concentrations (20-101 µM). OPA and MPA concentrations for these experiments were 4mM and 12mM, respectively

20 µM 30 min 35 min 40 min 45 min 50 min 55 min

0.141 0.144 0.146 0.147 0.147 0.147

40 µM

60 µM

80 µM

101 µM

Slope (AU/uM)

Intercept

0.270 0.276 0.279 0.280 0.281 0.282

0.406 0.413 0.416 0.417 0.418 0.418

0.532 0.541 0.545 0.548 0.549 0.55

0.671 0.681 0.687 0.690 0.691 0.692

0.0066 0.0067 0.0067 0.0067 0.0068 0.0068

0.0016 0.004 0.0057 0.0066 0.0073 0.0085

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R2 0.9998 0.9998 0.9998 0.9998 0.9999 0.9999

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Table 2: Average molar extinction coefficients, absorptivity (cm-1 M-1) calculated for each amino acid, compared to values reported by Svedas et al.21, the ratio is calculated as the values from this study over the previously published absorptivity by Svedas, the relative standard deviation around the ratio was 9.8% Amino Acid

This study (cm-1M-1 )

Svedas21 (cm-1M-1 )

Ratio

Aspartic Acid

6522

4800

1.36

Threonine

5480

5200

1.05

Serine

7149

5500

1.30

Glutamic Acid

6915

4900

1.41

Glycine

7367

5000

1.47

Alanine

7170

5400

1.33

Valine

7115

5600

1.27

Methionine

7108

5200

1.37

Isoleucine

6183

5000

1.24

Leucine

6818

4800

1.42

Tyrosine

5162

5200

0.99

Phenylalanine

6811

5700

1.19

Tryptophan

6404

4700

1.36

Lysine

11462

8800

1.30

Histidine

6250

4800

1.30

Arginine

7235

5700

1.27

Average

6947

5394

1.29

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TOC graphic:

Absorbance

2

1

0 250

300

(nm)

350

400

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