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Research Article pubs.acs.org/journal/ascecg
Molecular and Lumped Products from Hydrothermal Liquefaction of Bovine Serum Albumin James D. Sheehan and Phillip E. Savage* Department of Chemical Engineering, 119 Greenberg Complex, The Pennsylvania State University, University Park, Pennsylvania 16802, United States S Supporting Information *
ABSTRACT: We examined the decomposition of and product formation from a model protein, bovine serum albumin (BSA), under hydrothermal conditions. BSA forms smaller polypeptides as it decomposes, primarily via hydrolytic cleavage of the peptide bonds. It also forms insoluble aggregates in high yields at mild conditions, which subsequently decompose. There were no polypeptides (MW > 5 kDa) remaining after 5 min at 350 °C, but smaller peptides persisted in yields of about 15 wt %. The total yield of free amino acids was typically 40 wt %) of biocrude tend to do so at the expense of both biocrude quality and recovery of N and P in the aqueous phase. Typically, biocrude produced at these temperatures contains 4 to 7 wt % N,18−21 which is much higher than that in petroleum crude oil (0−2 wt %).22,23 N in biocrude not only reduces the heating value of this fuel precursor, but it also diminishes the process sustainability by reducing the amount of N that can be recovered from the aqueous phase for recycle as nutrients.
Hydrothermally processing low-value, wet biomass feedstocks such as food waste,1,2 municipal sludge,3−5 livestock manure,6 and microalgae7 is a step toward producing fuels and chemicals from these renewable resources. The reaction environment of hot compressed water decomposes the macromolecular constituents of biomass into a mixture of chemical products that can include amino acids, fatty acids, potential nutrients (e.g., ammonia, nitrates, phosphates), fuel gases, and renewable crude bio-oils. Hydrothermal processes are attractive for valorizing wet biomass because they obviate an otherwise energy-intensive drying step generally required by other processes (e.g., pyrolysis, lipid extraction).7−9 Hydrothermal liquefaction (HTL) typically operates at temperatures around 280−370 °C and pressures of 10−22 MPa (below the critical temperature (374 °C) and pressure (22.1 MPa) of water) to transform biomass into an energydense crude bio-oil that has 70−95% of the energy density of petroleum crude oil.7 In addition, HTL of biomass forms other product fractions that include a nutrient-rich aqueous coproduct, gases consisting primarily of CO2, and a solid residue. Transforming biomass into biocrude via HTL is facilitated by the unique properties of hot compressed water as it approaches its critical point. Specifically, the dielectric constant decreases, allowing for an increased solubility of small organic compounds. The ion product increases by approximately 3 orders of magnitude, which increases the concentrations of © XXXX American Chemical Society
Received: August 18, 2017 Revised: September 18, 2017
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DOI: 10.1021/acssuschemeng.7b02854 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
Research Article
ACS Sustainable Chemistry & Engineering
Figure 1. Amino acid profiles of BSA (red) and the average of five microalgae (green) species reported by Becker.33 The error bars represent the standard deviation.
temperatures and times that include both “fast” HTL18 with low reaction times (∼60 s) and high heating rates (8−11 °C s−1) and the more conventional isothermal HTL with batch holding times up to an hour. This approach of examining HTL of a model protein over a wide range of reaction severities and from both molecular and operational perspectives breaks new ground regarding the chemical reaction paths in HTL and how best to engineer them for biocrude production and nutrient recovery.
N-containing compounds that partition into biocrude originate predominantly from the peptide bonds that link individual amino acids together in protein. During HTL, proteins hydrolyze into smaller peptides and amino acids, which can either further react to produce N-containing heterocycles that partition into biocrude9 or decompose into water-soluble amines, organic acids, and ammonia. The pathway to water-soluble products is desired because it facilitates nutrient recycling and increases the sustainability of HTL. Previous work on the HTL of protein-rich biomass17,20,24−27 has provided valuable information regarding optimum conditions for producing biocrude and recovering N in the aqueous-phase products. These studies focused primarily on operationally defined lumped product fractions, such as biocrude, aqueous-phase products, solid residues, and gaseous products, as opposed to molecular products. While such studies reveal the effects of process conditions on yields of lumped product fractions, they offer little revelation regarding the identity and abundance of molecular species that constitute these product fractions. There is another body of work dealing with the hydrothermal decomposition of proteinaceous waste1,2,28 and model proteins,29−32 typically with the aim of producing and recovering individual amino acids or peptides. These studies do provide details about molecular products, but they provide no information about the biocrude and aqueous lumped product fractions. There remains a large gap in the literature between the engineering oriented studies focused on producing biocrude and the fundamental molecular studies concentrating on hydrolysis of proteins. This work aims to fill that gap by providing molecular details about protein-derived reaction intermediates and their transformation into biocrude or aqueous-phase products under HTL conditions. We report on the decomposition of a well-characterized, water-soluble, model protein (bovine serum albumin (BSA)) in hot compressed water into molecular products such as smaller peptides, amino acids, amines, and ammonia. In addition, we determine the yields of biocrude. We explore a wide range of
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MATERIALS AND METHODS
Materials. Bovine serum albumin (Sigma-Aldrich) served as the model protein. Figure 1 compares the amino acid composition of BSA and the average of five microalgae species reported by Becker.33 BSA has less alanine (Ala) and glycine (Gly) and more glutamic acid/ glutamine (Glu) than microalgae; however, the other 15 amino acids are comparable in abundance. Deionized water prepared with a Direct-Q3 UV-R EMD Millipore system was used as the reaction medium. Dichloromethane (HR-GC grade, EMD Millipore) was used for recovering biocrude from the batch reactors. Acetonitrile (HPLC grade, BDH), tetrahydrofuran (HPLC grade, Fisher Chemical), and trifluoroacetic acid (TGI) were used to prepare mobile phases for high performance liquid chromatography (HPLC) analyses. Marfey’s Reagent (Thermo Scientific) and acetone (HPLC grade, EMD Millipore) were used to prepare a precolumn derivatization agent for reverse phase (RP) HPLC analysis of amino acids. All amino acid standards were of HPLC grade and purchased from Sigma-Aldrich. Pierce BCA Protein Assay Kits (Thermo Scientific) were used for determining peptide concentrations. Ninhydrin reagent (Sigma-Aldrich) along with Lleucine solutions with known concentrations was used for determining primary and secondary amine concentrations. Ammonia TNTplus Vial Tests (high range, HACH) were used for quantifying ammonia concentrations. Reactors and HTL Procedure. We assembled batch reactors (∼1.3 mL internal volume) from 316 stainless steel tubing (1/4 in. O.D., 12 cm length) and Swagelok stainless steel caps. The reactors were loaded with a carefully measured amount of premixed aqueoussolution containing 2.5 wt % BSA. Once loaded with BSA solution, the reactors were tightly sealed and placed into a preheated Techne IFB 51 fluidized sand bath set at 200, B
DOI: 10.1021/acssuschemeng.7b02854 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
Research Article
ACS Sustainable Chemistry & Engineering 250, 300, 350, or 400 °C for batch holding times ranging from 0.5 to 60 min. For subcritical set-point temperatures (T < 374 °C), the solution loading was such that the liquid would expand to fill the reactor upon reaching the set point temperatures. For experiments conducted at 400 °C, enough BSA solution was loaded into the reactors such that the pressure would be 400 bar at temperature. Upon reaching the desired holding times, the reactors were carefully removed from the sand bath and quenched in an ice water bath. Once cooled to room temperature, the exterior surfaces of the reactors were dried and then allowed to equilibrate at room temperature for 1 to 2 h. Proxy reactors were assembled similarly to the batch reactors except they contained a thermocouple (1/16 in. O.D., Model: TJ36-CAXL116U-18-CC-XSIB, Omega Engineering) and were loaded with only deionized water. The internal volumes of the proxy reactors were approximately 1.1 mL. The internal temperatures of the proxy reactors were recorded every 0.1 s with an Omega UWBT series wireless transmitter, and the Supporting Information provides the reactor temperature profiles. It would take 60 to 75 s for the internal temperature of the reactors to reach the set point temperature of the sand bath, and the average heating rates ranged from 2.9 to 5.0 °C s−1. Product Recovery and Analyses. After opening the reactors, their contents were transferred to their own respective test tubes. We then recovered additional materials from the reactors by washing with 6 mL of deionized water followed by 6 mL of dichloromethane, each in small aliquots. The washings were transferred to the test tube containing the original reactor contents. The test tubes were inverted several times to promote mixing between the aqueous and organic phases and thereafter centrifuged at 500 rcf for 5 min. Subsequently, the aqueous and dichloromethane phases were each carefully transferred via pipet to their own respective test tubes to await further analyses. The solid phase products remained in the original test tubes used for recovering the products from the reactors. Dichloromethane was evaporated from the biocrude by flowing N2 over the surface of the samples at 38 °C with a Labcono RapidEvap Vertex Evaporator, and the remaining dried residue was biocrude. The solid samples were dried for 12 h in an oven at 70 °C. The dried product fractions were weighed to quantify their gravimetric yields which were the mass of the dried product fraction divided by the mass of BSA loaded initially into the reactor. Additionally, a Shimadzu GCMS QP-2010 Ultra with an Agilent DB-5MS column (30 m by 0.25 μm) was used to separate and identify some of the individual molecules in the biocrude. Molecular species were tentatively identified by comparing their mass spectra against those stored in a NIST mass spectral database. The aqueous phase was analyzed for its concentrations of peptides (via Pierce BCA Protein assay kit), primary and secondary amines (via Ninhydrin reagent), and ammonia (via HACH TNT 832 Ammonia Kit), following standard protocols provided by the suppliers. In some previous work on the decomposition of biomass in hot compressed water,34−37 the ninhydrin reagent has been used in an attempt to quantify concentrations of only amino acids. However, these measurements might be inaccurate because ninhydrin interacts not just with amino acids but with many different primary and secondary amines.38 Therefore, we consider all measurements quantified via the ninhydrin reagent to include all molecular species containing primary or secondary amine groups. The absorbances for each test were measured with a Shimadzu UV-3600 spectrophotometer. The concentrations of peptides, ammonia, primary and secondary amines, and amino acids in the aqueous phase were determined via calibration curves formulated with standards of known concentrations. Select samples of the aqueous phase underwent ultrafiltration using Spin-X UF 5k MWCO concentrators (Corning). Compounds with molecular weights lower than 5 kDa could pass through the membrane filter, while those greater would not pass. Subsequently, the “filtrate” and “concentrate” portions of the aqueous-phase products were analyzed for peptide concentrations via the Pierce BCA protein assay kit. This measurement provides information about the molecular weights of the peptides in the aqueous phase. Additionally, the aqueous-phase products underwent a precolumn derivatization with
Marfey’s reagent (1-fluoro-2,4-dinitrophenyl-5-L-alanine amide) to identify species of amino acids. Marfey’s reagent covalently binds chromophores to amino acids, which promotes interactions with the nonpolar stationary phase of the HPLC column, leading to higher resolution and increased photometric detection.39 The free amino acid concentrations were measured by a Shimadzu UFLC XR equipped with a diode array detector operating at a wavelength of 340 nm. A binary gradient mobile phase method was implemented and a LiChrospher R8 5 μ 10 Å column (30 cm × 4.6 mm) was used. The concentrations of aspartic acid and asparagine were reported collectively, as were the concentrations of glutamic acid and glutamine. Product yields were calculated as the mass of the product divided by the mass of BSA originally loaded into the reactors. The data presented in the following section are the average values from two or three runs, and the error bars are the sample standard deviations.
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RESULTS AND DISCUSSION This section presents the yields of peptides, amino acids, primary and secondary amines, ammonia, biocrude, and solids and how their respective yields vary with temperature and time. Additionally, identities of several molecular species in biocrude are provided. To the best of our knowledge, this represents the first report of such detailed molecular information combined with product fraction yields from HTL of protein. As such, it provides a new opportunity to take a step toward linking the actual chemistry with desired macroscopic outcomes such as biocrude production and nutrient recovery. Peptides. Figure 2 shows the temporal variation of the yields of peptides with molecular weights exceeding 5 kDa at
Figure 2. Yields of polypeptides (MW > 5 kDa) from HTL of BSA at different set point temperatures.
each set point temperature. For the balance of this article, peptides with molecular weights exceeding 5 kDa are referred to as “polypeptides”, while those less than 5 kDa are referred to as “peptides”. BSA has a molecular weight of approximately 66.5 kDa. Note that Figure 2 and all subsequent plots showing temporal variation use a log scale for the x-axis. This transformation is done so that the reader can better see the significant changes in yields that often take place at short reaction times, while the reactors are heating to their set point temperature. For all experimental conditions, the polypeptide yields decreased rapidly during HTL for the first minute. At the set point temperatures of 300, 350, and 400 °C, the yields of polypeptides decreased to about 30 wt % after only 30 s. For temperatures exceeding 250 °C, the yields of polypeptides decreased further with increasing holding time until they were C
DOI: 10.1021/acssuschemeng.7b02854 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
Research Article
ACS Sustainable Chemistry & Engineering entirely depleted. At 200 °C, the yields of polypeptides decreased to 13 wt % at 1 min but subsequently increased to approximately 40 wt % at longer holding times. The reemergence of polypeptides is likely due to the decomposition of water-insoluble BSA aggregates that formed earlier. Support for this hypothesis will be provided in the subsequent section regarding the formation of solids. Figure 3 presents the temporal variations of the yields of peptides. At set point temperatures ranging from 200 to 300
formation of solids to BSA denaturing and aggregating into water- and dichloromethane-insoluble materials. Aggregation of BSA occurs at temperatures exceeding 60 °C40 and has been documented previously41 during treatment of BSA in subcritical water. At 200 °C, for times exceeding 1 min, the yields of solids eventually decreased to 7.5 wt % at 60 min. The decomposition of solids coincides with the evolution of polypeptides at 200 °C for holding times exceeding 1 min, as noted previously. For temperatures exceeding 200 °C, a similar though less dramatic trend was observed wherein the yields of solids increased for holding times less than 1 min and then decreased at longer holding times. The lower yields of solids observed for short holding times (t < 1 min) at the higher temperature conditions (T ≥ 250 °C) may suggest that the heating rate of the hydrothermal medium affects the rate of BSA aggregation. Aida et al.31 recently reported on the effects of heating rate on the hydrothermal decomposition of BSA. The authors compared the decomposition pathways of BSA in a flow reactor with high heating rates (130−180 K s−1) to a batch reactor with slow heating rates (0.25 K s−1). They proposed that proteins decomposed under a random scission mechanism at high heating rates, whereas at slow heating rates the proteins aggregate and hydrolysis takes place at the solid−liquid interface. The results of the present study, which show a reduction of solids at higher set point temperatures (higher heating rates), are consistent with this hypothesis. Amino Acids. Figure 5 displays the temporal variation of the total yields of amino acids (sum of yields of all 18 detected
Figure 3. Yields of peptides (MW < 5 kDa) from HTL of BSA at different set point temperatures.
°C, the yields of peptides generally increased with increasing temperature and holding time. The highest yields of peptides were approximately 20 wt %, observed at set point temperatures of 350 and 400 °C for 1 min. At these higher temperatures, once the yields of peptides reached their maxima, they declined steadily to approximately 8 wt % at 60 min. Overall, at set point temperatures exceeding 250 °C, the coinciding decomposition of polypeptides (Figure 2) and the modest yields of peptides (
