Aqueous Fiber Spinning From a Renewable Protein Waste Material

Thursday, October 20, 2011: 2:30 PM
L100 B (Minneapolis Convention Center)
Jason W. Soares1, Steven Arcidiacono1, Charlene M. Mello1, Madhusudan Vasudevamurthy2 and Juliet Gerrard2, (1)U.S. Army Natick Soldier Research Development and Engineering Center, Natick, MA, (2)The University of Canterbury, Christchurch, New Zealand

Crystallin proteins from animal eye lenses represent a renewable, environmentally responsible biopolymer source for bio-derived fibers.  The crystallin protein family is a diverse and highly conserved set of proteins that form the macromolecular structure of the lenses of many vertebrates.  Despite variability in lens anatomy across organisms, these proteins are consistently organized in a supramolecular structure that is optically clear, preserving the ability of the lens to refract light.  Due to their natural functions, these proteins possess a range of interesting features which make them attractive for the production of new biomaterials such as self-assembly and the formation of stable, well ordered structures.  These structures include soluble protein, nanofibrils, and amorphous aggregates, each derived from native conformations.  Here, we describe the aqueous processing and spinning of crystallin proteins generated from a waste material, namely fish eye lens, into durable fibers.  Crystallin protein was isolated from North Atlantic Haddock (Melanogrammus aeglefinus) eye lenses through a simple homogenization technique.  Intact lenses were extracted and gently stirred at room temperature in an aqueous homogenization buffer designed to minimize protein aggregation and stabilize intermediate assembly states.  After 24 hrs, the fish lens homogenate (FLH) is clarified by centrifugation and collected as an opaque suspension in which crystallin protein is present in both amorphous and structured forms.  The FLH is comprised of a series of related structural proteins, which are used for subsequent investigations without further purification. 

Aqueous spinning of fibers from the FLH entails a delicate balance of processing, spinning and post-spinning variables.  The desired aqueous processing approach creates a uniform, soluble protein solution removing insoluble aggregates.  The processing approach must stabilize solution dynamics to facilitate proper ordering and organization of the protein, at a molecular level.  Minimization or suppression of aggregation during solution preparation and aging at relatively high concentrations is required to generate a reproducible spin solution for fiber spinning.  Toward designing suitable processing conditions, FLH was dialyzed into biological buffers and concentrated through ultrafiltration.  Several variables throughout the process were considered including buffer composition, protein concentration and spin solution aging.  Dialysis buffer composition was varied by supplementation with a number of amino acid stabilizers (i.e. aggregation suppressors) at varying concentrations including glycine, lysine and arginine. Buffer exchange was also conducted under varying temperature and pH conditions.  While glycine and lysine enhanced protein solubility as amino acid concentration increased, the influence on protein molecular assembly was not sufficient since gelation occurred relatively rapidly during spin solution aging.  However, supplementation with arginine improved protein solubilization and stabilization, enhancing protein yields and allowing for the preparation of concentrated spin solutions (~300-400 mg/ml).  Arginine resulted in reproducible, stable solutions which, after sufficient aging, facilitated suitable protein assembly states for subsequent formation of intact, durable fibers.  Interestingly, the arginine supplemented buffer was basic in nature (pH = 10.5) whereas glycine and lysine buffers were more acidic (pH 5 and 5.2 respectively).  The influence of pH relative to the presence of arginine on protein solubilization and stabilization is still under investigation.  Optimal processing temperature was determined as 4oC. 

Spin solutions of various concentrations and stages of supramolecular assembly were spun into fibers through a patented aqueous spinning approach.  The spinning technique was inspired by nature and, more specifically, the process by which a spider spins native dragline silk.  The design of the spinneret is based on the gradually tapered gland within the spider, which causes a shear-induced transition of self-assembled silk protein into a fiber.  Our lab-scale technique has previously been demonstrated in spinning recombinant spider silk proteins into fibers with mechanical integrity equivalent to natural dragline silk.  Spin solutions are loaded into the micro-spinneret, which has a volume capacity of 500 ul although typical spin trials are 50-75 ul.  A syringe pump applies a constant force at a constant rate (2-10 ul/min) to force the spin dope through PEEK tubing with I.D. ranging from 0.15-0.25 mm into a coagulation bath.  The fibers are allowed to coagulate within the bath and are collected after removal from the coagulation bath and submersion into a water bath.  Numerous spinning and post-spinning variables must be considered to optimize fiber formation, durability and mechanical integrity from concentrated spin dopes.  Here, this spinning technique was used to generate fibers from FLH solutions containing arginine at various protein concentrations and stages of spin solution aging, which correlate to various intermediate assembly states.  Fiber spinning variables considered include coagulation bath composition and spinneret I.D. while post-spinning variables include coagulation time and draw ratio.  Coagulation tests were conducted to choose optimal coagulation conditions for the various spin solutions.  Fibers up to 12 inches were generated although typically fibers ranged from 2-3 inches in length.  Fibers were of sufficient durability to withstand not only natural draw due to surface tension while removing from the coagulation bath but also extension to twice the original length prior to collection.  Drawn fibers were, on average, ~17 um in diameter while undrawn fibers were ~28 um.  Light microscopy of the as-spun fibers under white light was conducted to investigate surface and core defects along the length of the fiber.  Molecular alignment along the fiber axis was analyzed by investigating the birefringent nature of the single fibers under polarizing light with a first order red plate at 530 nm.  In general, fibers exhibited birefringence, which was indicative of some level of molecular order.  However, the fibers did contain a variety of defects that limited subsequent mechanical testing. 

Analysis of a compilation of spin trials enabled the identification of integral processing, spinning and post-spinning variables essential for formation of fibers from crystallin proteins.  Spin solution aging is dependent on protein concentration - higher concentration requires less aging to reach the appropriate conditions to spin fibers.  Increasing draw decreased fiber diameter, but molecular order was not enhanced at the current extensibility (~2-fold).  Optimal coagulation time was determined as 15-20 min for formation of the most durable fibers.  The information derived is being considered for optimization of processing and spinning processes to produce fibers with enhanced mechanical integrity.  Also under investigation is the use of nano-reinforcements, specifically nanofibrils, to enhance fiber properties.  Crystallin proteins readily form nanofibrils in solution, with branching and length being controlled by processing conditions and temperature.  Nanofibrils occur by subjecting the proteins to mildly destabilizing conditions that allow the proteins to partially unfold and refold.  Recent advances in our lab enable the control of nanofibril structure and morphology (e.g. length, bundling and branching).  Nanofibrils of up to 10 um in length with varying degrees of bundling and branching have been created and may serve as an additive that could further facilitate formation of fibers with increasing mechanical integrity from concentrated soluble protein spin solutions.  Alternatively, fibers can be spun directly from solutions containing nanofibrils. 

Fibers from crystallin proteins are a naturally-derived biomaterial that could be used in composite or textile applications including new fiber reinforcing material for (bio)composites or as a substitute for Nylon and polypropylene-based textiles.  Furthermore, the fibers represent a chemically diverse (defined by the amino acid side chains) platform for the future design of new multifunctional materials for use in military and civilian applications.  This work represents a significant advancement in the state-of-the-art regarding spinning of bio-derived fibers from a renewable waste protein material and offers a basis for spinning a range of biopolymers into useful materials.


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