METHODS FOR PREPARING TUNICATE DERIVED NANOCRYSTALLINE CELLULOSE, AND USES THEREOF
20250297037 ยท 2025-09-25
Inventors
Cpc classification
C08B15/08
CHEMISTRY; METALLURGY
C08B15/02
CHEMISTRY; METALLURGY
C09J197/02
CHEMISTRY; METALLURGY
International classification
C08B15/08
CHEMISTRY; METALLURGY
Abstract
A process is described for preparation of tunicate derived CNCs (T-CNCs) which exhibit a high aspect ratio, increased crystallinity and superior thermal properties compared to wood pulp derived CNCs (W-CNCs). The process enables scalable isolation of T-CNCs from tunicates, and a solution to the challenge invasive tunicates pose to aquaculture communities.
Claims
1. A method for preparing tunicate derived nanocrystalline cellulose (tCNC), comprising: collecting of or providing raw tunicate biomaterial; fibrillating the tunicate biomaterial to form a crude tunic pulp; deproteinating the crude tunic pulp under alkaline conditions with heating to solubilize said proteins, and then bleaching the deproteinated pulp; separating the deproteinated tunic pulp from reaction solution by hot filtering; adjusting pH of the filtered deproteinated tunic pulp to acid conditions, to produce a solid deproteinated and bleached tunic pulp, optionally with additional washing and hot filtering; fibrillating the deproteinated and bleached tunic pulp to produce a wet cellulose pulp base material; and hydrolyzing the wet cellulose pulp base material with a strong acid to produce said t-CNC.
2. The method according to claim 1, wherein the method further comprises a step of separating internal organs from the tunic of the raw tunicate biomaterial prior to said fibrillating; optionally wherein said separating step comprises mechanically pressing the raw tunicate biomaterial using a pressing device to loosen the tunic-organ connection within the tunicates.
3. The method of claim 2, wherein the step of separating internal organs from the tunic of the raw tunicate biomaterial comprises: pressing the raw tunicate biomaterial prior to said fibrillating using a pressing device to rupture the tunic and loosen the connection with organ material; and optionally physically washing the pressed tunicate biomaterial; stirring the pressed tunicate biomaterial in water for a time and at a speed effective to separate the tunic form the internal organs; screening the stirred, pressed tunicate biomaterial to remove the water and produce separated tunic and organ components; and collecting the tunic.
4. The method according to claim 1, wherein the separated tunic is fibrillated using a grinding mill, garburator or woodchipper.
5. The method according to claim 1, wherein the crude tunic pulp is deproteinated using an alkaline solution of NaOH, KOH or a mixture thereof and heating at from 50-75 C. for 1 to 24 hours, followed by said bleaching.
6. The method according to claim 5, wherein the alkaline solution comprises about 1.0-10.0 wt % NaOH, KOH or a mixture thereof.
7. (canceled)
8. The method according to claim 5, wherein the alkaline solution comprises about 4.0-5.0 wt % NaOH, KOH or a mixture thereof.
9. The method according to claim 1, wherein bleaching the deproteinated pulp is carried out by adding a bleach solution containing NaOCl with between 5 and 15% active chlorine and acetic acid at a concentration between 5 wt. % and 97 wt. %.
10. The method according to claim 1, wherein the hot filtering comprises filtering the deproteinated tunic pulp one or more times over a fiberglass screen mesh reinforced with a metal screen mesh.
11. The method according to claim 1, wherein the acid conditions are at or below pH 3.0.
12. The method according to claim 11, wherein the pH is adjusted using a strong acid.
13. The method according to claim 12 wherein the strong acid is sulfuric acid or hydrochloric acid.
14. The method according to claim 12 wherein the strong acid is at a concentration range of 1-10 wt. % acid.
15. The method according to claim 1, wherein the fibrillating of the deproteinated and bleached tunic pulp is carried out using a grinding mill or a garburator, to produce a fine pulp.
16. The method according to claim 15, wherein the fine pulp is a homogenous material where individual fibers can no longer be visually distinguished in the tunicate cellulose pulp.
17. The method according to claim 1, wherein the hydrolyzing comprises adding the strong acid to the wet cellulose pulp base material, mixing, quenching to neutralize the strong acid, allowing the resulting t-CNC to settle in solution, washing the settled solid t-CNC material, and then concentrating the washed T-CNCs to a final product.
18. Tunicate derived nanocrystalline cellulose (tCNC) prepared according to the method of claim 1.
19. A coating comprising a tunicate derived nanocrystalline cellulose (tCNC) prepared according to the method of claim 1.
20. An adhesive comprising a tunicate derived nanocrystalline cellulose (tCNC) prepared according to the method of claim 1.
21. A packaging material comprising a tunicate derived nanocrystalline cellulose (tCNC) prepared according to the method of claim 1.
22. (canceled)
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] The aspects, features and advantages of the invention will become more readily apparent from the following description, reference being made to the accompanying drawings in which:
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DETAILED DESCRIPTION
[0056] Described herein are methods for preparing tunicate derived nanocrystalline cellulose, and uses of the nanocrystalline cellulose materials derived from these methods. The methods described involve collection of tunicates, pre-processing, and post processing resulting in T-CNC. Other valuable co-products are also obtained during the process, including but not limited to tunicate derived proteins.
[0057] Tunicates are marine animals which contain highly pure cellulose in their tunic, the unique leather-like epidermis of the animal from which its name is derived. This tunicin cellulose may be hydrolyzed with appropriate procedures to yield T-CNCs, which possess among the highest aspect ratio and crystallinity of all known CNC sources. Current commercial W-CNCs have an aspect ratio of 10-20 and tend to display lower crystallinity (60-80%) than T-CNCs, which possess an aspect ratio of 50-100 and crystallinity commonly exceeding 90%. The potential advantages of a widely available CNC source, possessing both high crystallinity and high aspect ratio, are broad in scope. However, T-CNCs are only isolated at lab scale currently and, as a result, most recent research focuses on commercially available W-CNCs.
[0058] As a further incentive, tunicates are an invasive nuisance species causing economic challenges for the local aquaculture community on Prince Edward Island. The present invention therefore provides a means to directly address the growing problems invasive tunicates cause, turning nuisance tunicate species into a valuable resource, utilized to the benefit of the local aquaculture community and economy.
[0059] Higher aspect ratio CNCs lead to improved stress transfer in composites, reduced concentrations necessary for gelation, and enhanced viscosity modification. Moreover, T-CNCs can be used in combination with W-CNCs to form hybrid CNC mixtures which possess broad and tailorable aspect ratio distributions. These hybrid CNC mixtures lead to the enhancement of all in-plane and some out-of-plane mechanical properties in hybrid CNC films. Such hybrid mixtures can, in certain embodiments, enhance stiffness in polymer composites compared to individual CNC sources.
[0060] Previous attempts to isolate T-CNCs first involve the removal of non-cellulose tunicate components via manual separation, alkaline and bleaching pretreatments. This is then followed by treatment of the purified cellulose to yield CNCs with varying surface chemistries. Non-cellulose components are generally removed using ether moderate temperature, standard pressure, chemical treatments; or by using more mild chemical treatments combined with increased temperatures and pressures.
[0061] In prior work, the inventors utilized a three step hydrothermal treatment to isolate and compare T-CNCs from numerous tunicate species at lab scale. While highly pure T-CNCs were isolated in a reasonable yield by this method; the necessity of a sealed pressure vessel at elevated temperatures over multiple processing steps limited the scalability of this process. For these reasons, chemical pretreatments performed at moderate temperatures and standard pressure were selected for the presently described method, to develop more scalable methods for tunicate pretreatment.
[0062] Once the purified tunicate cellulose is obtained, it can be surface modified using numerous approaches, such as but not limited to 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) mediated oxidation and sulfuric acid hydrolysis, or left unmodified using hydrochloric acid hydrolysis. The most common of these treatments for both wood and tunicate derived CNCs is sulfuric acid hydrolysis. Under appropriate conditions, this results in the nearly complete hydrolysis of amorphous cellulose content to yield CNCs, and the concurrent grafting of negatively charged sulfate groups to the CNC surface. These charged groups reduce interactions between neighboring CNCs, limiting the agglomeration and flocculation of CNC suspensions and allowing for their dispersion in a wider range of solvents.
[0063] The high aspect ratio of T-CNCs make them more susceptible to agglomeration and flocculation than other comparatively low aspect ratio W-CNC sources. This motivated the inventors to design the described large-scale T-CNC isolation process to yield sulfated T-CNCs.
[0064] After hydrolysis is complete, the acidic CNC solution is typically quenched followed by salt removal and concentration of the aqueous CNC suspension. In lab scale CNC isolation, a combination of conventional filtration techniques, centrifugation, and dialysis are commonly employed to obtain a purified and concentrated CNC product. These techniques are limited in scalability, challenging to replicate or optimize, and often result in significant loss and/or contamination due to multiple small-volume product transfers. This has led to the adoption of highly scalable tangential flow filtration (TFF) systems. In TFF, the feed flows tangential to the membrane, leading to a continual defouling of the membrane surface by the feed components. This allows for the large scale diafiltration and subsequent concentration of CNCs in a single system, leading to increased efficiency and reduced loss from product transfers. In this study, TFF was utilized to both purify and concentrate the isolated T-CNCs, demonstrating that this scalable technique can be applied to T-CNCs.
[0065] The primary challenges of scaling up T-CNC isolation historically have been either a lack of available tunicates, difficulties in the large-scale harvesting of tunicates and the limited amount of available literature surrounding T-CNC isolation, at any scale. Some of the challenges associated with T-CNC isolation may be mitigated by both unique local factors and by the growing global effects of climate change. Recently, tunicates have been causing great concern to aquaculture industries in the Maritime Provinces of Atlantic Canada.
[0066] In the inventors' previous work, they highlighted how the mussel industry in Prince Edward Island (PEI) has been threatened by growing costs, reduced mussel harvests and the need to constantly apply anti-fouling treatments to fishing gear as a result of this tunicate infestation. Through Dynamic Energy Budget modelling, a recent study predicted that invasive tunicates may reduce mussel production by more than 20%.sup.217. This is highly relevant to the local economy as PEI harvests over 80% of all blue mussels sold in Canada, while also selling product internationally.sup.218,219. In PEI alone, there are four different tunicate species, all of which are invasive and of foreign origin.sup.108,215-217. As the climate warms, the conditions under which tunicates thrive become more prevalent, leading to increased tunicate densities and growing challenges for local aquaculture communities.sup.109,217,220-222.
[0067] However, it is demonstrated that: 1) the scalable harvesting of tunicates is possible and 2) high quality T-CNC can be isolated from these local invasive tunicates. The commercial scale harvesting of tunicates could directly address the challenges of high tunicate density in local waterways. Easing the burden on members of the aquaculture community by harvesting tunicates for scalable T-CNC production may lead to a shift in the perception of invasive tunicates from a destructive nuisance species, to that of an abundant and available resource to be harvested and utilized.sup.109. Local waters surrounding PEI, along with similar marine environments worldwide with dense tunicate populations, serve as accessible sources of tunicate feedstock, with potential for scalable T-CNC isolation.sup.223.
[0068] In the following Examples, the commercial scale extraction of high aspect ratio T-CNCs from the abundant feedstock of invasive tunicates on PEI is described and optimized and the inventors' experiences during the various steps, from harvesting to T-CNC isolation, are discussed. Various characterizations are performed to better understand the behavior and challenges of preparation as well as the attributes of the final T-CNCs. Experiences from large-scale preparation of W-CNCs using established protocols as well as the ultimate characteristics of W-CNCs and other nanocelluloses are described for comparison.
EXAMPLES
Preparation of a Tunicate-Derived Cellulose Feedstock
Harvesting
[0069] The starting material for the pilot-scale production of W-CNCs is a high-purity commercial cellulose pulp prepared by well-established wood pulping protocols. Obviously, the preparation of a similar cellulose feedstock from tunicates is necessarily a very different process. To prepare a relatively large quantity of tunicate cellulose feedstock, we began by manually harvesting approximately 20 kg of invasive Styela clava tunicates from waterways surrounding PEI. Manual harvesting is a viable process to collect commercial scale quantities of tunicates. In fact, it is estimated that over a million pounds of Styela clava (wet weight) are cultivated and harvested annually from waters around South Korea, where they are consumed as a seafood delicacy known locally as mideuduck.sup.107,225,226. These have primarily been manual tunicate harvesting methods similar to those employed here. Although we posit efficient automated processes may lower harvesting costs. Recently, Ocean Bergen AS implemented an automated approach for harvesting tunicates from Norwegian waters to extract protein for animal feed 223.
[0070] The processing of the tunicates after harvesting is schematically shown in
Tunic Preparation
[0071] Once harvested, the cellulose-containing tunics were manually separated from the protein-rich internal organs. We are currently investigating more economically viable approaches including automated tunic separation and a biorefinery-type approach which utilizes the entire tunicate as a process input. The manually prepared tunics used here were washed, dried and ground as described in Supplemental Materials Section S1. While others have used the internal organs to prepare animal feed 106 or to ferment bioethanol 39, we chose to focus on T-CNC isolation and simply disposed of the internal organs. The use of such byproducts is left for future work. Generally, one half of a tunicates weight is its tunic, although this varies with tunicate species, environmental factors and life cycle stage. We found that our 20 kg of Styela clava tunicates harvested from PEI waters resulted in an estimated 10 kg of tunic, which were 90% water, yielding approximately 1 kg of dried tunic powder.
Tunic Pretreatment
[0072] To isolate T-CNCs from this tunic powder, the cellulose must be purified, and the non-cellulose components removed to prepare a high cellulose feedstock for acid hydrolysis. To accomplish this, the tunic powder was shipped to the Forest Products Laboratory where it was further processed by alkaline deproteination treatments and bleaching following the protocols described by van den Berg et al., with modifications as described in Supplemental Materials Section S1. The overall yield for the deproteination and bleaching steps was 31%, comparable to the yields reported in Table 1 for similar processes at lab scale. The final bleached material was used as the feedstock for preparing T-CNCs by acid hydrolysis.
TABLE-US-00001 TABLE 1 Other reports of tunicate cellulose purification strategies and respective yields. Pretreatment Tunicate Feedstock Deproteination Bleaching Yield (%)* Reference Salpa fusiformis Pronase in buffer NaOH reflux N/A .sup.227 Styela clava KOH NaClO/CH.sub.3COOH N/A .sup.7, 136, 145, 207 Halocynthia roretzi KOH NaClO/CH.sub.3COOH N/A .sup.228 Halocynthia NaOH NaClO.sub.2 N/A .sup.210 papillosa Halocynthia roretzi NaOH NaClO.sub.2 N/A .sup.229 Halocynthia roretzi NaOH H.sub.2O.sub.2 20 .sup.211, 230, 231 Drasche Styela clava NaOH NaClO.sub.2/CH.sub.3COOH 31 This Work Styela clava Three step hydrothermal process 40 .sup.49 Ciona intestinalis Prehydrolysis (H.sub.2SO.sub.4) 30 .sup.49 Ciona intestinalis Kraft Cooking (NaOH/Na.sub.2S) 12 .sup.9, 224 Ascidia sp. Bleaching (NaClO) 3 .sup.9, 224 Cynthia roretzi Step 1 & 2: 180 C. | 2 h 30 .sup.111 Styela plicata Step 3: 75 C. | 1 h 24 .sup.9, 224 Halocynthia roretzi 21 .sup.9, 224 *Approximate values | N/A = Not Reported.
[0073] According to Zhao and Li, generally tunic possesses a 50:50 weight ratio of carbohydrates to proteins, where between 75% and 95% of the carbohydrate fraction is glucose, and of the glucose fraction, between 50% and 75% is cellulose.sup.9. Although their work focuses on four different tunicate species, we feel that their general conclusions are applicable to our processing. Therefore, this suggests that the 1 kg of dried tunic powder prepared for this work likely possesses only 19-36% cellulose. Given this estimate, coupled with the findings reported in Table 14, our overall yield of 31% for the deproteination and bleaching steps seems reasonable.
[0074] While the additional non-cellulose tunicate components present a challenge when isolating T-CNCs, these additional components have intrinsic value and may be recoverable. Although not the focus of this study, we suggest that additional value-added product streams, including protein 232 and heavy metal recovery (see Supplemental Materials Section S2).sup.233-236, may be feasible if tunicates are processed to T-CNC in a biorefinery-type approach. This requires thoroughly understanding the components of waste streams generated in T-CNC isolation and determining their recoverability, an active area of investigation in our group.
CNC Preparation
[0075] Wood derived W-CNCs are prepared from high purity cellulose wood pulp (97% cellulose) in the Nanocellulose Pilot Plant at the Forest Product Laboratory using standard protocols.sup.38. The main steps in the process are: 1) sulfuric acid hydrolysis, 2) diafiltration to remove by-products, and 3) concentration of the resulting aqueous CNC suspension. Tunicate derived T-CNCs were prepared similarly, albeit on a smaller scale, and with necessary changes to accommodate differences in the source materials. Our experiences during the various steps of the T-CNC preparation are discussed below along with relevant comparisons to W-CNC processing and proposed changes to protocols that may improve the process.
Sulfuric Acid Hydrolysis
[0076] Hydrolysis of the tunicate cellulose was accomplished using 64% H.sub.2SO.sub.4 for 2 hours with additional details described in Supplemental Materials Section S1. The hydrolysis yield was 42% for T-CNCs, compared to 50% for the optimized W-CNC isolation, resulting in aspect ratios of 65 and 12 respectively (
TABLE-US-00002 TABLE 2 Other reports of CNC isolation with varying production scale and hydrolysis conditions. Cellulose Production H.sub.2S0.sub.4 CNC Aspect Hydrolysis Source Scale Hydrolysis Ratio* Yield (%)* Reference MCC .sup.# Lab (64%, 2 h) 15 30% .sup.237 Cotton Lab (65%, 30 min) 12 N/A .sup.12 Cotton Lab (60%, 4 h) 15 N/A .sup.231 Wood Lab (64%, 25 min) 28 33% .sup.3, 113 Wood Lab (64%, 2 h) 23 N/A .sup.7 Wood Pilot (64%, 1.5 h) 12 50% This Work & .sup.38 Wood Commercial (64%, N/A) 20 N/A .sup.203, 238 Tunicate Lab (60%, 1.5 h) 50 N/A .sup.207 Tunicate Lab (65%, 2 h) 72 N/A .sup.230 Tunicate Lab (60%, 32 h) 70 N/A .sup.231 Tunicate Lab (50%, 20 h) 63 N/A .sup.229 Tunicate Lab (48%, 13 h) 70 N/A .sup.12 Tunicate Lab (48%, 3 h) 100 N/A .sup.7 Tunicate Lab (55%, 20 min) 35 30% .sup.224 Tunicate Lab (50%, 4.5 h) 80 50% .sup.239 Tunicate Pre-pilot (64%, 2 h) 65 42% This Work *Approximate values | N/A = Not Reported | .sup.# = Microcrystalline Cellulose (MCC)
[0077] In many reports, information such as yield, and precise processing conditions unfortunately are omitted. However, we note that the T-CNCs prepared here display properties consistent with previous T-CNCs isolated at laboratory scale. Indicating that the impressive properties attributed to T-CNCs can, as pioneered in the development of large-scale W-CNC isolation, be preserved when T-CNC isolation is scaled up. At this time, replicate experiments and concurrent process optimization of T-CNC isolation at this scale remain future areas of study. Also, as discussed later, some material was lost during diafiltration, which adversely affected the T-CNC yield. Therefore, with further improvement of protocols, the T-CNC yield could very well approach that of the W-CNCs.
Diafiltration and Concentration
[0078] Following hydrolysis, the reaction is quenched and neutralized with aqueous NaOH. The resulting highly saline suspension leads to the association and settling of CNCs. Most hydrolysis by-products could then be removed by decanting the supernatant, adding deionized water, again allowing CNCs to settle and repeating the process. Eventually as salinity decreased the CNCs began to suspend rather than settle, and a tubular ultrafiltration unit was used to complete by-product removal by tangential (cross) flow filtration (TFF).sup.38. TFF reduces filter cake formation by creating turbulent flow, which improves flux rate compared to conventional dead-end filtration (See
[0079] The make-up water was then shut off to concentrate the CNC suspension until its viscosity increase inhibited flow through the membranes tubes, after which the system was back flushed to yield the concentrated CNCs. The CNC suspension viscosity is primarily governed by the aspect ratio of the CNCs and the CNC concentration, where the salinity of the suspension is assumed to be consistent since both W-CNCs and T-CNCs are neutralized prior to filtration.
[0080] As observed previously by others.sup.178,242,243, high aspect ratio CNC suspensions display considerably higher viscosity values than lower aspect ratio CNC suspensions. In our case, the maximum concentration of the high aspect ratio T-CNC was 1.3 wt %. This is far below the 10 wt % achievable with lower aspect ratio W-CNCs, but similar to that found for TEMPO pretreated wood-derived cellulose nanofibrils 244.
Further Processing Protocol Steps and Embodiments
[0081] Further embodiments of the described methods may include, from extraction of the tunicates to isolation of the T-CNC, keeping the cellulose containing material wet as drying should be avoided to prevent hornification. Hornification results from the formation of hydrogen bonded networks during drying that are only partially reversible. Here, we initially dried the tunic and between each process step the yield was determined by drying the intermediate product. This may have led to a compounding of the hornification of cellulosic material, resulting in reduced efficacy of chemical treatments and a lower process yield. If drying is necessary, lyophilization is preferable to air or oven drying to lessen the effect of hornification. Traces of color were still observed after the bleaching step, which we attempted to remedy with additional bleaching after acid hydrolysis. To obtain whiter and cleaner materials, alternating between acid chlorite bleaching and alkaline extraction may be a beneficial procedural improvement. The level of calcium in the final T-CNC product is quite high at 0.054 wt %, as typical levels observed in W-CNC processing are less than 0.002 wt % (See Section S2 of Supplemental Materials). We expect that the source is likely the tunicates' natural calcium-rich environment. The T-CNC hydrolysis is highly acidic and when the reaction was neutralized, it likely caused association of the negatively charged CNC sulfate groups with calcium cations. The calcium level may be reduced by the addition of an acid wash after bleaching, by decanting the acidic T-CNC solution after hydrolysis but before neutralization, or with suitable chelation treatments. After the hydrolysis and neutralization, some aggregates were observed which interfered with subsequent ultrafiltration and purification steps. Simple screening for these aggregates prior to filtration may improve ultrafiltration efficacy. With scaling, it may also be advantageous to replace the large-scale centrifuging, if it is still found necessary after other improvements, with mechanical homogenization to improve yield, processing efficiency and overall consistency of the T-CNC product. Given the very different size distributions of the two types of CNCs, it may be possible to increase the T-CNC concentration efficiency by optimizing the pore size of the ultrafiltration membranes.
CNC Properties
[0082] Once the T-CNCs and W-CNCs were prepared and their morphologies understood, we compared their crystallinity and thermal stabilities while contrasting our findings with past reports. What follows is our assessment of the results and how the properties of the obtained T-CNCs compare to that of W-CNCs prepared by an optimized process.
Crystallinity
[0083] We assessed the overall structural order of as produced CNCs utilizing two complimentary techniques: XRD and Raman. A summary of our findings and those reported by others for CNCs prepared by similar procedures is displayed in Table 16 and
TABLE-US-00003 TABLE 32 Steady shear viscosity of 1 wt % T-CNC and W-CNC suspensions (a) steady shear and (b) small amplitude oscillatory (SAOS) data for the T-CNC suspension. Cellulose 380- 93- source XRD* NMR Raman Raman Reference MCC 84 NA 77 68 .sup.237, 246 Cotton 92 75 77 68 .sup.229, 246 Wood 80-89, 90, 72 60 56 46 .sup.113, 156, 246 Tunicate 91, 95 80, 94 70 93 .sup.7, 246 Wood 66, NA 56 46 This work Tunicate 75 NA 68 96 This work NA is not available | *For wood, wide range attributable to varying methods used to calculate crystallinity.
XRD
[0084] The overall structural order of the prepared CNCs was further assessed by calculating their percent crystallinity from the background-corrected experimental diffractograms in
Raman
[0085] Raman crystallinities of the wood and tunicate CNCs were determined using two methods380-Raman (Agarwal et al. 2010; 2013) and 93-Raman (Agarwal et al. 2018), and the values are reported in Table 16. The methods are based, respectively, on the band intensity ratios 380/1096 cm.sup.1 and 93/1096 cm.sup.1 in the Raman spectra of the CNCs visible in
Thermal Stability
[0086] To understand the thermal stability of the T-CNCs isolated in this work and how it compares to W-CNCs, we performed TGA in both an oxidizing (air) and an inert (nitrogen) environment. The resulting thermograms and their derivatives were obtained and compared with those of W-CNC analyzed in the same manner. As visualized in
[0087] The onset of thermal degradation for W-CNCs is clearly lower (
TABLE-US-00004 TABLE 43 Reported oxidative thermal properties of various CNCs prepared by H.sub.2S0.sub.4 hydrolysis. Cellulose Onset Inflection H.sub.2S0.sub.4 Source Temperature* Point* Hydrolysis Reference Wood 235 253 (64%, 2 h) .sup.237 Wood 210 240, 421 (64%, 1.5 h) This Work & .sup.38 Wood 255 275 (64%, N/A) .sup.238 Tunicate 180 N/A (50%, 3 h) .sup.248 Tunicate 180 N/A (50%, 4.5 h) .sup.239 Tunicate 106# 128# (60%, 20 min) .sup.249 Tunicate 290 N/A (55%, 1 h) .sup.208 Tunicate 190 200 (55%, 20 min) .sup.224 Tunicate 225 291 (64%, 2 h) This Work *Approximate values | N/ANot Reported | #Measured in an inert environment.
Perspective and Outlook
[0088] By processing roughly 20 kg of invasive tunicates to H.sub.2SO.sub.4 hydrolyzed T-CNCs, this work accomplishes the largest scale isolation of T-CNCs reported to date. Learning from the pilot scale development of W-CNCs, we isolated T-CNCs using scalable techniques, with reasonable yield, and of similar properties to those reported for T-CNCs isolated at laboratory scale by others. The overall yield of our pretreatment (31%) and acid hydrolysis (42%) of the tunic powder was within the range of values reported for laboratory scale tunicate to T-CNC processes. Overall, the yield of T-CNCs from our process was 12.2% based on the dry weight of the tunic powder and T-CNCs isolated therefrom. Experimentally determined aspect ratios, crystallinity, and some thermal properties of the T-CNCs exceeded those of W-CNCs, as expected; and were similar to those found for T-CNCs prepared at laboratory scale by others. Replicate trials that implement the numerous potential process improvements described here would likely lead to a considerable increase in yield and quality of T-CNCs at this scale, and we feel that the proposed improvements themselves are scalable in nature. Other procedures for T-CNC isolation may be scalable and modified from literature in a likewise manner. These may yield T-CNCs of similar or improved properties based on the process conditions, tunicate source and degree of process optimization.
[0089] We posit that the commercial scale isolation of tunicate derived CNC is feasible and that the unique properties of these T-CNCs, which complement the growing global utilization of nanocellulose materials, justifies this pursuit. We chose an invasive species negatively affecting local aquaculture communities in PEI and across Atlantic Canada as the T-CNC source. This allows us to demonstrate the unique conditions that currently exist on PEI, which mitigate the historic challenges of tunicate harvesting and T-CNC isolation at commercial scale. These conditions are not limited to Atlantic Canada and entities around the globe are currently harvesting tunicates at commercial scale for their proteinaceous components. Regardless of the driving force, tunicates will ultimately be considered and perhaps utilized as a large-scale source of numerous value-added products, including their unique animal-derived high aspect ratio cellulose, for commercial T-CNC isolation. This study lays tangible groundwork towards that goal, directly demonstrating the feasibility and results of kilogram scale tunicates to T-CNC processing, and promoting the widespread utilization of both invasive and native tunicates to produce useful and sustainable materials for the benefit of our growing global community.
Methods
[0090] A more detailed description of the T-CNC and W-CNC isolation process is provided in Section S1 Supplemental Materials. What follows are general descriptions of the equipment and techniques utilized to obtain the reported experimental data and is complimented further in the Supplementary Information.
Elemental Analysis
[0091] The sulfur, sodium and calcium content of the prepared CNCs was determined using Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES) (Ultima II, Horiba Jobin-Yvon, Edison, NJ, USA) using previously developed protocols.sup.155.
[0092] The qualitative elemental composition of the dried tunic powder used to prepare the T-CNCs was investigated with a JEOL JSM6400 Digital SEM, using the equipped EDX (Genesis) Energy Dispersive X-ray system. Digital X-ray maps were obtained from powdered samples which were mounted to carbon tape and carbon coated for conductivity prior to imaging.
Transmission Electron Microscopy (TEM)
[0093] To assess the morphology of the T-CNC and W-CNC, transmission electron microscopy (TEM) micrographs were obtained on a JEOL 2011 STEM instrument. Dilute (0.001 wt %) colloidal suspensions were cast onto etched copper coated grids and air-dried prior to imaging. The average length, width and aspect ratio were calculated from at least 50 measurements from 5-10 representative micrographs of each sample using Image J software.
Fourier-Transform Infrared Spectrometry (FTIR)
[0094] Attenuated total reflectance Fourier transform infrared spectrometry (ATR-FTIR) was performed to understand the functional groups present, screen for impurities and to calculate the Lateral Order Index (LOI) and Total Crystallinity Index (TCI) of the isolated T-CNCs and W-CNCs. A Bruker Alpha FTIR spectrometer (Alpha-P) was utilized with OPUS software, 32 scans were averaged against background scans to yield the reported spectra in the range of 4000 to 500 cm 1. The measured transmittance values were converted to absorbance and the magnitude of the absorbance at 2900, 1430, 1375 and 897 cm.sup.1 was used to determine LOI and TCI.
Thermogravimetric Analysis (TGA)
[0095] Thermal properties were assessed with the aid of Thermogravimetric analysis (TGA) which yielded thermal decomposition profiles for T-NCC and W-CNCs, as well as their first derivative with respect to weight (DTGA) thermograms. Experiments were performed on a TA Instruments TGA Q500 under an oxidizing atmosphere (60 mL/min compressed air, 40 mL/min nitrogen) from room temperature to 700 C., using a heating rate of 10 C./min. Inert atmosphere thermograms were obtained by first purging the sealed sample chamber for 30 minutes with a 100 mL/min nitrogen flow rate, after which the sample was heated at 10 C./min to 700 C. under nitrogen.
X-Ray Diffraction (XRD)
[0096] X-ray diffraction (XRD) was performed to assess the crystallinity of the isolated T-CNC and W-CNC used in this work. Aqueous CNC samples (0.5 wt %) were flash frozen in liquid nitrogen prior to lyophilization to obtain the dry CNC sample for analysis. The utilized Bruker AXS D8 Advance instrument was equipped with a graphite monochromator, variable divergence slit, variable anti-scatter slit and a scintillation detector. Cu (Ka) was the radiation source used (k=1.542 A) and the measurements were performed on glass slides with a double-sided scotch tape adhesive, in air, at room temperature, from 2-60 (20).
Raman Spectroscopy
[0097] For estimations of crystallinity by Raman spectroscopy methods (380-Raman and 93-Raman.sup.250-252), sample pellets were prepared with a pellet-forming die. Approximately 100 mg of T-CNCs and W-CNCs were used for making pellets. The CNCs were analyzed with a Bruker (Billerica, MA) MultiRam equipped with a 1064-nm 1,000 mW continuous wave (CW) diode pumped Nd:YAG laser. Spectra were recorded from 2,048 co-added scans using 600 mW laser excitation, as reported previously.sup.253.
[0098] In all cases, Bruker OPUS 7.2 software was used to process the spectral data which involved normalization, selection of a spectral region, background correction, and band integration. Background correction was performed using a 64 points OPUS rubberband option. For plotting purposes, the spectra were converted to ASCII format and exported to Excel.
[0099] CNCs crystallinity was estimated using two Raman methods380-Raman.sup.250,251 and 93-Raman.sup.252. The following two equations were used to estimate these crystallinities.
Rheometry
[0100] A stress-controlled Anton Paar rheometer (MCR 502) was used to carry out the rheological measurements at 25 C. Couette and double-Couette flow geometries were used for different samples. The region of linear viscoelasticity was first determined by performing strain-sweep tests. The viscoelastic behavior of the suspensions was determined from frequency sweep tests in the linear regime. The steady shear test was performed from low to high shear rate. The reproducibility of all data was investigated by repeating the tests three times. To eliminate the history effect, all samples were pre-sheared at shear of 100 s.sup.1 for 5 min followed by 30 min rest prior to all subsequent tests. To assure homogeneity of the suspensions and eliminate aging effect all the samples were ultrasonicated using a Sonics & Materials VCX500 probe, operating at 20 kHz, at a power of 60 W and energy of 10,000 J/g.sub.CNC, operated in pulses with suspensions placed in an ice bath to avoid overheating.
Larger Scale Isolation of Tunicate CNC [100 kg]
[0101] Lessons learned processing the 25 kg of raw tunicates to CNC described in above are applied in this example, where an improved process which isolates CNC from 100 kg of raw tunicate input is described. More attention is paid here to the large-scale collection and preprocessing of tunicates. This larger process, in which the cellulose is never dried, demonstrates clear progress towards directly addressing the tunicate problem on PEI.
[0102] In order to further demonstrate that T-CNC can be isolated from local invasive tunicates at large scale on PEI, we have processed 100 kg batches of locally sourced tunicates to T-CNC. This section describes a working iteration of this process which is based on our experience gained from lab scale T-CNC isolation at UPEI (20 g of raw tunicate input) and from larger scale T-CNC isolation at the Forest Product Laboratory (25 kg of raw tunicate input). Notably, this chapter describes the collection of raw tunicates and the necessary preprocessing steps which have been developed to facilitate the large-scale harvesting and standardization of the tunicate input. To our best knowledge this work represents the largest scale on which T-CNC has ever been isolated from tunicates.
Process Overview
[0103] Based on our prior experiences processing local invasive tunicates to T-CNC we felt that it makes the most sense to divide the process into the general steps outlined in the
Raw Tunicate Input
[0104] The first step in converting invasive tunicates to T-CNC is the collection of the tunicates, a process which results in the collection of significant non-tunicate material as a consequence of the tunicates natural environment and the method of tunicate collection. We collect our tunicates manually from fishing gear owned by Prince Edward Aqua Farms, however an automated process is under development. The tunicates were harvested at both Malpeque Bay and Montaque Bay locations, between the months of July to November. The process for washing this raw tunicate input and separating the tunicate from the non-tunicate material is currently accomplished manually, however the throughput is quite high (>50 kg/hour) due to the obvious visual differences between the tunicate and non-tunicate components. Examples of material removed in this manner are crabs, mussels, fishing gear (rope etc.), general waste (plastic packaging, construction material, etc.), and many other non-tunicate materials. The quantity of non-tunicate material in each batch of raw tunicate input, after manual sorting and washing with 50 L of fresh water is estimated at 20%, therefore 100 kg of raw tunicate input yields 80 kg of tunicate after non-tunicate removal and washing.
Tunicate to Tunic Pulp
[0105] After standardizing the tunicate input by cleaning and removing all visible contaminants, the next major step is to separate the protein rich internal organs from the cellulose rich tunic. At lab scale this is accomplished manually using a scalpel, however this is not a very efficient technique at larger scale. Therefore, we developed a more scalable technique for separating tunic from the organs using a low-cost combination of off the shelf and manual techniques described in the flow chart shown in
[0106] The tunicates were first squished by counter rotating rollers whose distance from each other was less than a quarter inch, the forces experienced by the tunicate when between the rollers ruptured the tunic and loosened the tunic-organ connection within the tunicates. These weakened tunicates are then immersed in a 200 L polyethylene barrel which is filled with fresh water to 100 L total volume. A standard spiral type of paint stirrer with ribbon design is mounted to a drill and is immersed into the aqueous dispersion, then the drill is turned on for 10 minutes. As an alternative method, a HP submersible sewage pump equipped with an 8-foot 2 inch diameter hose can be used to accomplish the same effect by circulating the entire solution for a similar period of time. The rotating paint stirrer (or pump) causes an aggressive mixing process which loosens and eventually separates the tunic form the internal organs in the aqueous medium. The entire contents of the barrel are then drained through a large, elevated screen which removes the water and results in clearly separated tunic and organ components on the screen. The screen is than manually cleaned with tunic and organs collected separately. At this point the tunic has been separated from the internal organs but the tunic is still largely intact. To make the tunic a more consistent input for later processing we fibrillate the tunic further to form a crude tunic pulp. Initial trials accomplished the tunic fibrillation to tunic pulp using a household garburator (a sink mounted waste disposal unit), however we also demonstrated this using a 13 HP woodchipper. Both processes produced a suitable tunic pulp for downstream processing however the woodchipper is a more robust and durable choice for this quantity of tunic input. The fibrillated tunic pulp is then collected and used as an input for the next step. A typical yield of wet tunic from a given quantity of wet tunicate is roughly 50%, therefore the 80 kg of tunicate input to this step results in 40 kg of tunic with the remainder being internal organ.
Tunic Pulp to Deproteinated Tunic Pulp
[0107] The fibrillated tunic pulp is then combined with NaOH in a 200 L polyethylene barrel with 2 kg of NaOH and enough water to reach 40 L of total volume. The barrel, containing an alkaline solution 4.5 wt % NaOH, is then heated using a blanket heater to 65 C. and left overnight (12 h). This process solubilizes the remaining proteins, lipids and some non-glucose sugars. At this point a base-bleach (chlorite bleaching under basic conditions) is performed by adding a 4 L bottle of household bleach (Giant Tiger, 5% active chlorine). The volume of the barrel is then immediately increased to 100 L with warm DI water, the blanket heater is set to 65 C. and the reaction is left overnight (12 h). This base bleaching makes use of the differing species which common household bleach forms under alkaline conditions compared to acidic conditions. As seen in
[0108] While this additional non-cellulose value-added product is not the direct focus of this report, we were able to use this tunicate soap throughout the remaining stages of tunicate processing. It was primarily used as a hand soap and as a cleaner for the processing equipment (plastic barrels, etc.); but it was also used as a way to demonstrate, particularly to the general public, that additional non-cellulose value-added products are possible with this tunicate to CNC process. In this way tunicate soap has allowed us to improve the sustainability of our processing and to connect more effectively with the general public and the policy members who represent them.
[0109] Once the deproteinated tunic pulp has been hot filtered from the reaction solution the barrel is flipped to face up and 100 L of warm water is added with manual agitation, once added the barrel is inverted and the product is again hot filtered. One more addition of 100 L is added before the final hot filtration (
Deproteinated Tunic Pulp to Bleached Tunic Pulp
[0110] The deproteinated tunic pulp prepared in the previous step is placed in a 200 L polyethylene barrel, the total volume of the barrel containing the filtered deproteinated tunic pulp is adjusted to 40 L by adding warm water. The pH is lowered to below 2 by adding 1 L of 98 wt. % sulfuric acid with the warm water and manually agitating for 5 minutes. A blanket heater is applied to the barrel and set to 65 C., after which point a bleaching charge of 250 mL of NaOCl (15% active chlorine) and 50 mL of 97% CH.sub.3COOH is added to begin the acidic bleaching process, and the reaction is left overnight (12 h). As an alternative, we have demonstrated that the 250 mL of NaOCl (15% active chlorine) and 50 mL of 97% CH.sub.3COOH bleaching charge can be supplemented by using 1 L of household bleach (Giant Tiger, 5% active chlorine) and 1 L of household vinegar (5% CH.sub.3COOH) instead.
[0111] The next day a second bleaching charge is added with manual agitation, and, at one-hour intervals, two additional bleaching charges are added with manual agitation. The barrel is allowed to settle for one hour following the addition of the final bleaching charge. After this point the solution is hot filtered, and the liquid and solid components are separated as in the previous deproteination step. The liquid components can be reused for neutralizing basic solutions or low-quality bleaching; however it is primarily disposed of at this point. Future work may reveal more uses for the liquid product of this step.
[0112] The solid component is the deproteinated and bleached tunic pulp, which is then washed and hot filtered with two additions of 100 L of warm water, as done in the previous step. The deproteinated and bleached tunic pulp is then fibrillated, using either a 5-inch diameter dish grinding mill or a household garburator, to a fine pulp (
Bleached Tunic Pulp to T-CNC Products
[0113] The never dried, deproteinated, and bleached tunic pulp is then hydrolyzed to tunicate CNCs which can be used in various downstream T-CNC products. The acid hydrolysis utilizes 45% H.sub.2SO.sub.4 (aq.) and is performed at a solid to liquid ratio of 1:10, premeasured acid is added to the wet cellulose pulp of known solid content and the dispersion is vigorously mixed for 2 hours at 45 C. Due to safety concerns in handling large volumes of concentrated acid in a confined laboratory setting, we hydrolyzed an aliquot of the tunicate cellulose pulp prepared in the prior step and have used the product of this smaller hydrolysis in subsequent steps. An aliquot of 150 grams of never dried, deproteinated, and bleached tunic pulp was used in this hydrolysis step. This pulp was assumed to be entirely tunicate cellulose, and the solid content of the pulp was determined gravimetrically to be 10 wt % (15 grams of dry tunicate cellulose). The tunicate cellulose pulp was then adjusted to 20 wt % by evaporative drying and the resulting 75 grams of cellulose pulp was combined with 75 grams of 81 wt % H.sub.2SO.sub.4 (aq). After mixing the aliquot reaction for 2 hours at 45 C., the reaction was quench by pouring it into 2.5 L of reverse osmosis (RO) water. A mixture containing 60 grams of NaOH, and 2.5 L of RO water was then slowly added to the quenched aliquot reaction to neutralize the acid. This highly saline dispersion was then allowed to settle overnight, after which 4 L of supernatant was decanted from the settled and saline tunicate CNC dispersion. The remaining 1 L containing the saline aggregated tunicate CNCs was then placed in a tangential flow filtration (TFF) system, and an additional 10 L of RO water was passed through the TFF system to wash out residual salts. The T-CNC product was then concentrated to 670 grams of 1.0 wt % T-CNC, equivalent to 6.7 grams of dry T-CNC, a 45% yield of T-CNC based on the dry weight of the hydrolysis input (
[0114] After hydrolysis, the acidic reaction is quenched by diluting it with RO water (volume goes from 200 mL to 2.5 L), and neutralizing with dilute 5% NaOH (aq) to a final volume of 5 L. This highly saline dispersion is left overnight (12 h) which allows the settling of the T-CNC, about 4 L of the saline solution is removed by decanting the supernatant from the settled dispersion. The remaining 1 L containing the salty T-CNCs was diluted to 5 L total volume with RO water and additional salt was washed out by passing an additional 10 L of RO water through the dispersion using a TFF system. By continuing to run the TFF system without adding additional RO water, we were then able to then concentrate the washed T-CNCs to a gel like final product with 1.1 wt % T-CNCs. These CNCs can then be used in various downstream T-CNC applications.
Mass Flow of Process
[0115] The general mass flow of the tunicate to T-CNC process is displayed in
[0116] The total process for isolating tunicate CNC from 100 kg batches of raw PEI tunicates consumes 700 L of fresh water, 500 L of RO water, 6.5 kg of NaOH, 8 L Household Bleach (5% active chlorine) OR 2 L of NaOCl (15% active chlorine), 4 L Household Vinegar (5% CH.sub.3COOH) OR 250 mL of 97% CH.sub.3COOH, and 4.5 kg of (98%) H.sub.2SO.sub.4 (aq).
Energy Flow of Process
[0117] The energy flow for the process of isolating tunicate CNC from 100 kg batches of PEI tunicates is also provided (
[0118] The total energy used in this process was calculated from a summation of the energy used in each process step, to be 142.9 MJ. Future work could focus on understanding the energy consumption of various process steps and optimize the energy use.
Summary of Process Improvements
[0119] The major process improvements implemented in the 100 kg batch process for isolating T-CNC from local invasive tunicates are as follows:
[0120] Never dry the material (all the way from tunicate to T-CNC).
[0121] Developed a process to efficiently wash 100 kg batches of tunicates.
[0122] Developed a mechanical process to separate kilograms of tunic from tunicate guts.
[0123] Utilized 200 L plastic barrels and a hot filtering technique in the processing.
[0124] Utilized household equipment in process such as a grinding mill, or alternatively a garburator or wood chipper.
[0125] Implemented a base bleaching step to produce a less colored product.
[0126] Used household vinegar and bleach rather than lab grade CH.sub.3COOH and NaOCl.
[0127] Demonstrated the use of TFF to collect T-CNCs on PEI for the first time.
[0128] Together these improvements and the additional findings herein related to the isolation of T-CNC from invasive PEI tunicates, represent significant new knowledge creation in this area and bring us closer than ever to directly addressing the challenges local tunicates pose to PEI aquaculture.
A Note on Club Vs Vase Tunicates
[0129] Both club and vase tunicates were used throughout this work to isolate T-CNCs. While our initial lab scale T-CNC isolation made use of both species and compared the subtle differences between the CNCs isolated from both species; it was decided that it is better to focus on the more significant differences found between T-CNCs and commercially available W-CNCs. Therefore, the focus of the research regarding T-CNC isolation was to develop a scalable process which could be used to isolated T-CNCs from both club and vase tunicates. The focus was on increasing the scale of the process rather than comparing the respective yields and properties of the T-CNC isolated from both species at the same process scale. This is what justified the decision to use only club tunicates in the 25 kg batch processing, and to compare the resulting T-CNC with commercially available W-CNCs. Due to this decision the research was focused towards developing a working process for isolating T-CNC from 100 kg batches of tunicates, rather than spent on a comparative analysis of the properties and yield of T-CNC isolated from both species at the 25 kg batch scale. Notably, focusing on the comparison of T-CNCs to W-CNCs also provides useful context for the findings herein regarding hybrid W-CNC and T-CNC combinations.
[0130] Due to the support of Prince Edward Aqua Farms, we were able to procure 100 kg of vase and club tunicates to be used in our 100 kg T-CNC isolation process. However, the numbers reported herein are only for the process which used 100 kg of vase tunicates as a process input. The reason that numbers for club tunicates are unavailable at this time is that the author used the 100 kg of procured club tunicates as testing material to develop the numerous process improvements described in the previous sections. As a result, the club tunicates were consumed in a plethora of different experiments, trying various process iterations, which were crucial to the development of the working 100 kg batch process described herein. However, this exhausted the available 100 kg supply of club tunicates in the lab. Therefore, the process has, so far, only been successfully used to isolate T-CNC from 100 kg of vase tunicate input. However, future work to complete this same process with 100 kg of club tunicate input is feasible. Future work could be developed to optimize and compare the process for both species and to potentially scale up the process further.
Conclusions
[0131] The major conclusions of this work are listed below:
[0132] Local invasive club and vase tunicates both contain high quality T-CNC, a renewable and unique high aspect ratio nanomaterial, indicating that they are a valuable untapped resource with potential to be utilized for the benefit of PEI.
[0133] A batch wise process utilizing up to 100 kg of raw tunicate input to produce T-CNC has been demonstrated, indicating commercial scale isolation of value-added products such as T-CNC from local invasive tunicates is feasible.
[0134] The yield of T-CNCs from our processes was between 10% and 15% based on the dry weight of the tunic powder and T-CNCs isolated therefrom.
[0135] The T-CNC product isolated from local vase and club tunicates were demonstrated to have a superior aspect ratio to commercially available W-CNC. The T-CNC also displays superior thermal properties and a higher overall crystallinity than W-CNC.
[0136] As a result of its high aspect ratio, the T-CNC percolates a polymer matrix, changing its properties, at lower loading than commercially available low aspect ratio W-CNCs.
[0137] Hybrid mixtures of T-CNC and W-CNC have shown synergistic properties, and some of these properties seem to extend to CNC reinforced polymer nanocomposites.
[0138] This hybrid effect has been demonstrated in cast film samples utilizing hydrophilic PVA and hydrophobic PLA as polymer matrices.
[0139] The hybrid effect was not observed in PLA composites prepared by melt mixing. Although, due to severe aggregation of the hydrophobically modified CNCs, it is feasible that the hybrid effect could be observed in melt mixed composites if aggregation of the CNC can be reduced.
Applications for Tunicate CNC in the Global CNC Market.
[0140] The nanocellulose market is continuing to expand as society moves away from non-renewable products toward sustainable and renewable resources. The market for CNCs is currently dominated by W-CNCs derived from the pulp and paper industry. While these processes are efficient and afford a low cost W-CNC product, their low aspect ratio limits the potential applications for this CNC source. Tunicates by contrast yield CNCs with a much higher aspect ratio, however they are currently only made in small quantities and the economics of the processing is not fully understood. Interestingly, the concept of hybrid CNC mixtures provides a new opportunity in this area. By mixing T-CNCs with W-CNCs, these hybrid CNC mixtures can be made to have not only tailorable average aspect ratios, but also tailorable aspect ratio distributions. This allows for a family of hybrid CNC products to be prepared with tunable properties which are currently not achievable with W-CNC alone. Moreover, given the diverse nature of CNC applications, this family of tunable hybrid CNC products has potential to open up new areas which are currently unavailable to W-CNC due to limitations arising from its low aspect ratio. In general terms: mixing T-CNC and W-CNC results in a hybrid CNC product that has the potential to improve the properties of, and expand the applications for, W-CNC products. This research provides groundwork for development in this area, however future work is necessary to carry it forward.
Utilizing CNC Production to Address Local Invasive Tunicates on PEI.
[0141] By practically demonstrating the feasibility of large-scale tunicate CNC isolation on PEI, we highlight a direct solution to the problems invasive tunicates are causing local aquaculture. By developing other value-added products from the process such as tunicate soap or substituting laboratory grade acetic acid and sodium hypochlorite for vinegar and household bleach, we improve the feasibility of this process being developed commercially on PEI. Which has the potential to directly address the problems invasive tunicates pose while fostering the development of a new local bioeconomy centered on the collection of tunicates, their processing to value added products such as CNC, and the utilization of these local sustainable and renewable products in diverse applications.
Supplemental Materials:
S1-Processing
W-CNC Isolation
[0142] Wood-derived CNCs (W-CNCs) were prepared in the Nanocellulose Pilot Plant at the Forest Product Laboratory by sulfuric acid hydrolysis as previously reported (1). Briefly, sulfuric acid (64 wt %) at 45 C. was sprayed onto strips of prehydrolysis kraft rayon-grade dissolving wood pulp under nitrogen. The mixture was stirred at 45 C. for 90 minutes, after which the reaction was quenched with water. The suspension was then bleached with a hypochlorite solution followed by neutralization with NaOH. The W-CNC suspension was allowed to settle and the salt/sugar solution was decanted. The W-CNC suspension was then diluted such that the sodium sulfate concentration dropped to about 1 wt %, at which point the W-CNC particles began to disperse. The aqueous suspension was then transferred to a PCI Membranes A19 tubular ultrafiltration system equipped with FP200 tubular membranes (PVDF 200,000 MWCO) where, during circulation, the dilute salt/sugar solution passed through the membrane while W-CNCs were retained. Reverse osmosis (RO) water was added to maintain the W-CNC concentrate at 1 wt %. Diafiltration was continued until the residual salt concentration of the permeate was reduced to about 8 M, measured as a conductivity of 40-50 S/cm.sup.2. The dispersion was then concentrated to about 10 wt % solids using the tubular ultrafiltration system by circulating without replenishing the water. The overall yield was about 50%.
Tunicate Collection
[0143] Club tunicates (Styela clava) were collected from Malpeque Bay, Prince Edward Island, as described previously (2). Briefly, tunicates were collected and frozen for storage. The tunicates were then thawed and washed with deionized water to remove salt and other debris. The tunic was removed manually from the internal organs using a scalpel, and washed further with deionized water. The tunic was then dried and ground to a fine chalk-like powder using a T-Series 28,000 rpm Multi-function Grinder (HC-150 China) resulting in one kilogram of dried tunic powder (Table S1).
TABLE-US-00005 TABLE S4 T-CNC Isolation Process Yield Process Step 1: Step 2: Step 3: Input Deproteination Bleaching Hydrolysis Mass (g) ~1 Kg 386 284 118 Yield (%) 39 80 42
T-CNC Isolation
[0144] Isolation of T-CNCs from the tunic powder generally followed the process described by van den Berg et al. (3), with modification. The dried tunic powder was combined with 6 liters of 5 wt. % NaOH and mixed at 80 C. for 24 hours. The product was then filtered and washed with deionized water until the filtrate was below a pH of 10. This deproteinated tunic product was dried to determine solid content and then combined with deionized water and mixed at 60 C. The pH of the reaction was adjusted to 5.5 using glacial acetic acid. With strong stirring, 50 grams of 80% purity NaClO.sub.2 and 50 mL of glacial acetic acid were added to the reaction mixture. The reaction was covered and allowed to react for 1 hour at 60 C. with periodic stirring. Then a second addition of 50 grams NaClO.sub.2 and 50 mL of glacial acetic acid was added and stirred periodically for 1 hour. Three more additions of NaClO.sub.2 and glacial acetic acid were made in the same fashion. The product was then allowed to cool and settle overnight, followed by filtering, washing with deionized water and drying to determine solid content. Finally, the 280 g of bleached tunic powder was hydrolyzed to T-CNCs by adding five liters of 64 wt % H.sub.2SO.sub.4 with strong stirring for 2 hours at 45 C. The hydrolysis was then stopped by diluting with cool deionized water to a volume of 100 L. Because of residual color, an additional 10 grams of NaClO.sub.2 was added and the dilute T-CNC suspension was allowed to bleach for an additional hour, after which 10% NaOH was used to neutralize the acidic solution to a final volume of 200 L. This highly saline, dilute T-CNC suspension was allowed to settle overnight. Roughly 170 L of supernatant was then removed and the remaining 30 L containing the T-CNC was circulated through a tubular ultrafiltration system (PCI Membranes series flow 1.2 meter B1-modules) equipped with FP200 tubular membranes (PVDF 200,000 MWCO), a circulating pump (Grundfos CR-2), heat exchanger and holding tank. This was a similar setup as for the W-CNC preparation, only smaller.
[0145] Residual sodium sulfate was removed by diafiltration over 48 hours, after which the permeate conductivity was measured below 50 S/cm. During the initial filtration process, aggregated tunicate derivatives obstructed the circulation pump, reducing the operating efficiency of the equipment. We corrected this by cleaning the pump and centrifuging the suspension using a Sharples model AS16 centrifuge (17500 G, two minute retention time) to remove smaller aggregates (See S4). The aggregated tunicate derivatives were separated, dried, and were found to weigh <2 grams. However, this small amount of aggregates lead to additional processing and cleaning steps which lead to a further reduction of yield. We speculate that these aggregates are in fact the same areas of the tunic removed during the tunic powder preparation step performed by van den Berg et al. (3), which was described as a particularly difficult area of the tunic to deproteinate near the animals' siphons. In the lab scale, van den Berg et al. (3) removed these areas, however emulating commercial scale processing we used the entire tunic. After centrifugation, remaining aggregated tunicate derivatives were removed via filtration using an 80 mesh screen, leading to additional product loss. The diafiltration process was then completed and the T-CNC suspension was concentrated. A total of 118 g of T-CNCs were produced in this process as an aqueous suspension approximately 20 L in volume and 0.58 wt % concentration, representing a 42% yield for the H.sub.2SO.sub.4 hydrolysis.
CNC Dispersability
[0146] Both W-CNCs and T-CNCs are prepared as aqueous suspensions. To determine if these solutions could be dried and later redispersed; we lyophilized samples of both and attempted to redisperse them with the aid of sonication. As seen in
S2Elemental Analysis: ICP-OES and EDS
TABLE-US-00006 TABLE S2 Elemental analysis of T-CNCs and W-CNCs via ICP-OES. Sulfur Sodium Calcium T-CNCs 0.61 wt % 0.35 wt % 0.054 wt % WCNCs 1.06 wt % 0.87 wt % <0.002 wt % * * Based on typical values observed in past W-CNC isolation.
[0147] Measured EDS data for dried tunic powder is shown in
S4Dynamic Light Scattering (DLS)
Laser Light Scattering
TABLE-US-00007 TABLE S4a Dynamic Laser Light Scattering information obtained for aggregate laden T-CNC solution and small-scale initial T-CNC isolation following the van den Berg et al. procedure (3). Mean Effective Diameter (MED) Polydispersity Small Scale 395.6 nm (s.d. 6.81) 0.273 (s.d. 0.022) Crude T-CNC 908.4 nm (s.d. 68.6) 0.376 (s.d. 0.016) T-CNC Centrifuge 1X 675.9 nm (s.d. 68.3) 0.362 (s.d. 0.010) T-CNC Centrifuge + 8X 445.1 nm (s.d. 22.1) 0.329 (s.d. 0.019)
[0148] A Sharples model AS16 centrifuge (17500 G, two minute retention time) was used twice to remove small aggregates in combination with manual screening (80 mesh) for large aggregates. First, the T-CNC suspension was centrifuged 1 (2 minutes retention time) and screened for aggregates. Then the suspension was centrifuged again, and after 8 (16 minutes retention time) through the centrifuge, the MED and polydispersity were determined to be comparable to a small scale batch of T-CNCs (Table S4a) prepared by the procedure of van den Berg et al. (3). The T-CNC suspension was then screened a final time and the diafiltration and concentration processes were completed. These findings are in close agreement with similar reports of DLS measurements on T-CNCs isolated at lab scale (5). Although not shown, AFM was used to determine individualization of the T-CNCs produced by the small scale batch, ensuring it was a suitable qualitative standard for our larger scale processing.
TABLE-US-00008 TABLE S4b Zeta potential of the final W-CNC and T-CNC products. Sample W-CNC T-CNC Zeta potential (mV) 52.90 41.09
[0149] Zeta potential was performed using a Brookhaven NanoBrook 90PlusPALS system. The W-CNC and T-CNC samples were sonicated for 20 minutes, diluted with deionized water and were analyzed at 25 C. The total number of cycles for each sample was 30 with Inter-cycle delay of 1 second.
S5CNC Morphology
[0150] We note that both the W-CNCs and T-CNCs display relatively high average width (6, 7), which we attribute to incomplete hydrolysis, to the parallel clustering of CNCs during TEM analysis, to a combination of both, or to human error when obtaining and statistically analyzing the TEM images. Irrespective, a reduced CNC width results in an increasing CNC aspect ratio, indicating that our TEM analysis may, if anything, underestimate the aspect ratio of both CNC sources. The length and width distributions of W-CNC and T-CNCs are shown in
TABLE-US-00009 TABLE S5 Morphology of T-CNCs and W-CNCs determined from TEM W-CNCs T-CNCs Length 215 56 1214 505 Width 20 5 20 6 Aspect Ratio 12 4 65 25
S6FTIR
TABLE-US-00010 TABLE S6 Noted FTIR signals for W-CNC and T-CNC. Wavenumber (cm.sup.1) Functional Group Reference 3340 OH (8) 2900 CH (9) 1640 HOH (10, 11) 1160 COC (9, 12) 1050 COC (9, 12) 897 C.sub.1 (anomeric) (13)
[0151] The FTIR spectra of W-CNC and T-CNC are visible in Figure S6, and tabulated in Table S6. They display similar characteristic peaks associated with CNCs and cellulose more broadly. The broad absorption in the area around 3300 cm.sup.1 is indicative of OH stretching, arising from both intermolecular and intramolecular hydrogen bonding. Other expected signals are observed at 2900, 1160 and 1050 which result from CH symmetric stretching, COC glucose skeletal stretching and COC pyranose stretching, respectively (9, 12). A small signal visible at 1640 cm.sup.1 has been shown to represent free H.sub.2O (10, 14). Although the CNC samples were lyophilized to remove H.sub.2O, we assess that the highly hydrophilic nature of these materials led to H.sub.2O absorption from the environment during and immediately prior to analysis. Additionally, a peak associated with the anomeric (C1) vibration of the (1-4) glycosidic linkages present in cellulose was observed at 897 cm.sup.1 (13).
[0152] The Lateral Order Index (LOI) and Total Crystallinity Index (TCI) of the isolated T-CNCs and W-CNCs was assessed by comparing the spectral ratio of the 1430/897 cm.sup.1 (15, 16) and the 1375/2900 cm.sup.1 (17) bands respectively (
S7 XRD
[0153] Both CNC sources displayed intense signals characteristic of the 200 crystalline cellulose reflection and weaker signals associated the 004 cellulose reflection at 22.5 and 34.5 20 (11). The T-CNCs produced in this work display signals at 15 and 17 (2), consistent with the 1
TABLE-US-00011 TABLE S7 Summary of structural properties obtained from XRD diffractograms. Crystallographic Plane 1
S8TGA
[0154] Feng and Hsieh observed that a decrease in the thermal stability of CNCs is related to an increase in surface area as particle size decreases (20). Since the W-CNCs are smaller particles with a higher surface area than T-CNCs, this finding is consistent with our experimental results. The appearance of separate shoulders in the TGA thermograms of W-CNCs in air may be due to the presence of surface sulfate groups (11, 21). The elimination of H.sub.2SO.sub.4 from the sulfated CNCs requires little thermal energy, leading to a lower thermal stability of CNCs that are sulfated compared to unmodified CNCs (22). In this work however, both the T-CNCs and W-CNCs studied were modified with surface sulfate groups, yet only the W-CNCs exhibit multiple separate shoulders in TGA thermograms performed in air. Using ICP-OES (S2) we determined that the sulfur content of the W-CNCs is almost double that of the T-CNCs (1.06% and 0.61% respectively). While these are in the typical range for CNCs isolated via sulfuric acid hydrolysis from wood (23) and tunicate cellulosic feedstocks, the considerable differences in sulfur content complicate comparisons between W-CNC and T-CNC thermograms.
[0155] The TGA thermograms displayed changes in the rate of mass loss which are clearly visible in the derivative TGA (DTGA) thermograms. By observing the DTGA thermograms, the inflection points of the T-CNCs are clearly visible preceding that of the W-CNCs in air (
TABLE-US-00012 TABLE S8 Summary of thermal properties obtained from TGA and DTGA thermograms. TGA-Air TGA-Nitrogen T-CNC W-CNC T-CNC W-CNC Onset Temperature ( C.) 225 210 239 237 Ash (%) 3 4 8 19 DTGA-Air DTGA-Nitrogen T-CNC W-CNC T-CNC W-CNC Inflection Point ( C.) 291 240 & 421 300 295
NCC Based Coating for Improving the Properties of Paper Packaging
[0156] Paper based packaging could potentially replace plastic by being natural and biodegradable. However, the requirement for higher mechanical properties and physical properties, and cost of production could be a challenge. PEI Bag Co. produces paper bags for potato packaging. A master bag is a two-ply SOS style bag typically measuring 14 in7 in by 32 or 34 in long. The commodity items sold to local and the US customers accounting for a strong percentage of PEI bag Co sales. While the level of customer bag breakage is minimal, there are always issues with breakages. The PEI Bag corporation is interested to understands ways that can reduce the breakages of the bags.
[0157] In this project a biodegradable coating comprising of tunicate-based cellulose nanocrystals and polyvinyl alcohol is investigated to improve the mechanical and physico-chemical properties of the paper bags. The section below discusses the methods and the results for various compositions of the coating materials.
Materials & Methodology
[0158] To prepare the biodegradable glue, 5 g of the polyvinyl alcohol (PVA) pellets (MW=124 kDa) was mixed with 95 g of distilled water in a beaker. The mixture was placed on a heated plate at 90 C. with strong stirring for three hours to allow full dissolution of the PVA. Then the 5 wt % PVA solution was mixed with different proportions of 1 wt % tunicate derived CNC. The final glue compositions are represented in the table below. In samples 1-4, the total solid content of the aqueous glue solution is roughly 0.23 wt %, and in samples 5-8 the total solid content is roughly 0.46 wt %. Sample 9 is a control sample without any adhesives added. The total CNC content of the adhesive is also varied between 0 and 5 wt %, to determine the ideal CNC:PVA ratio necessary to optimize the desired mechanical performance characteristics of the paper samples.
TABLE-US-00013 PVA CNC Total solid Total solvent Sample Glue PVA (weight %) CNC (weight %) content of content of number applied (g) (grams) dry (grams) dry glue (wt %) glue (wt %) 1 10 0.25 100 0 0 0.25 99.75 2 10 0.2375 98.958 0.0025 1.041 0.24 99.76 3 10 0.225 97.826 0.005 2.173 0.23 99.77 4 10 0.2 95.238 0.01 4.761 0.21 99.79 5 10 0.5 100 0 0 0.5 99.5 6 10 0.475 98.958 0.005 1.041 0.48 99.52 7 10 0.45 97.826 0.01 2.173 0.46 99.54 8 10 0.4 95.232 0.02 4.761 0.42 99.58 9 0 0 0 0 0 0 0
[0159] After preparing the glue samples, 10 g of each prepared glue was applied equally between two layers of A4 size paper sheets cut from paper bags provided by PEI Bag Company. The samples were dried for 24 h under compression. After drying, the samples were sent to an independent laboratory operated by Georgia-Pacific for analysis.
Results & Discussion
Mechanical Properties
[0160] The tensile strength (TS) is described as the maximum tensile stress that a polymer can tolerate. Elongation at break (EB) is described as the maximum change in length of the test sample at the breaking point. Tensile strength (TS), and elongation at break (EB) were evaluated by various ASTM standard methods at the Georgia Pacific Lab. Three replicates samples were analyzed for each film specimen. TS, and EB were calculated by Eq. (1) and Eq. (2) respectively. Data was acquired for both samples oriented parallel to the machine direction (MD) and for samples oriented perpendicular to (or cross) the machine direction (CD).
[0161] Where FMax is maximum load, AMin is minimum cross-section area, LMax is extension at the moment of rupture, L0 is the initial length of the specimen.
[0162] The mechanical properties of the samples were assessed and the resulting data is presented in Table 2. As expected the values for MD were higher than the CD values. In terms of the tensile strength, comparison between the treated samples and the control sample showed a significant difference. In addition to this, the tear strength of the treated samples was higher than the control sample which confirms the increase in mechanical properties of the paper product. Generally speaking it is clear that introducing an additional PVA:CNC adhesive between the paper layers has led to significant mechanical improvements. The PVA on its own provides significant support, however this is primarily a viscous reinforcement prone to deform under strain, leading to the enhanced % Elongation observed in the MD samples. To balance the viscous nature of the PVA, CNC is added to increase the elastic component of the adhesive. This translates to the enhanced Tensile, Tear, and Wet Tensile reinforcement observed in samples with elastic CNCs reinforcing the viscous PVA. Overall, the mechanical properties of all samples were improved by the addition of PVA:CNC adhesive and the CNCs are clearly providing a tunable elastic reinforcement to the adhesive.
[0163] To prepare Sample 3, a total of 0.2375 g of PVA, 0.0025 g of CNC and 9.76 g of deionized water was used to laminate two standard A4 sheets of paper (Surface Area 0.0625 m.sup.2), resulting in significant mechanical performance improvements. Extrapolating this out allows us to estimate that 3.8 g of PVA, 0.04 g of CNC and 156.16 g of water while be necessary to laminate 1 square meter of paper in the same manner. Based on paper bags measuring 14/7/34 (Surface area 1526 in2 or 0.9845 m2), the material used to fully laminate a single double ply paper bag would be 3.74 g of PVA, 0.039 g CNC and 153.7 g water. Assuming negligible water cost and a market price of PVA ($3/kg in bulk [Alibaba]) and CNC (10$/kg in bulk [CelluForce]); this translates to an estimated additional cost of 1.162 cents to laminate each bag with a fully biodegradable adhesive of the described composition. If this represents 60% of the overall cost, then total cost including equipment and other cost for implementing it could be 2 cents. It is anticipated that the adhesives prepared for this study can be further optimized and that the estimated additional cost per bag could either be slightly lowered while preserving mechanical properties, or, for a moderate increase in cost per bag, additional additives could be utilized in an effort to further enhance mechanical performance.
TABLE-US-00014 Tensile, Tensile, Elongation, Elongation, Wet Tensile, Wet Tensile, Tear Tear Sample CD MD CD MD CD MD CD MD number (MPa) (MPa) (%) (%) (Mpa) (Mpa) (N) (N) 1 261 468 7.4 2.1 71 135 1.24 1.34 2 289 483 8.0 1.8 75 148 1.36 1.42 3 283 479 7.2 1.8 73 138 1.50 1.40 4 296 483 8.5 1.5 71 136 1.63 1.25 5 295 331 7.7 1.9 69 147 1.37 1.67 6 288 493 8.5 1.3 73 146 1.55 1.39 7 246 485 6.9 2.4 67 137 1.59 1.26 8 240 387 7.3 1.9 71 75 1.61 1.25 9 (control) 131 213 7.9 1.3 34 55 1.03 1.14
Conclusions
[0164] The results of the study showed that the application of fully biodegradable CNC-based glue enhanced the mechanical properties of the paper samples. By tuning the ratio of PVA:CNC we can control the mechanical performance characteristics of a laminated paper product. Adhesive samples were demonstrated to improve the Tensile, Wet Tensile and Tear resistance of all samples in both the MD and CD directions compared to samples without adhesive. Samples containing PVA reinforced with CNC (Sample 3 for example) displayed further improvements in the Tensile, Wet Tensile and Tear resistance compared to the neat PVA reinforcement. Using Sample 3 as an example, we estimated an additional adhesive cost of roughly 1.2 cents per bag based on the current market prices for the bulk materials. We assess that this adhesive, or one of similar composition, has the capacity to be applied on the paper bag production line for a practical scaled-up study.
[0165] While illustrative and presently preferred embodiments of the invention have been described in detail hereinabove, it is to be understood that the inventive concepts may be otherwise variously embodied and employed and that the appended claims are intended to be construed to include such variations except insofar as limited by the prior art.
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