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Protein/CaCO3/Chitin Nanofiber Complex Prepared from Crab Shells by Simple Mechanical Treatment and Its Effect on Plant Growth Yihun Fantahun Aklog 1 , Mayumi Egusa 2 , Hironori Kaminaka 2 , Hironori Izawa 1 , Minoru Morimoto 1 , Hiroyuki Saimoto 1 and Shinsuke Ifuku 1, * 1

2

*

Graduate School of Engineering, Tottori University, 4-101 Koyama-Cho Minami, Tottori 680-8552, Japan; [email protected] (Y.F.A.); [email protected] (H.I.); [email protected] (M.M.); [email protected] (H.S.) Faculty of Agriculture, Tottori University, 4-101 Koyama-Cho Minami, Tottori 680-8553, Japan; [email protected] (M.E.); [email protected] (H.K.) Correspondence: [email protected]; Tel.: +81-857-31-5592

Academic Editor: Hitoshi Sashiwa Received: 1 August 2016; Accepted: 14 September 2016; Published: 22 September 2016

Abstract: A protein/CaCO3 /chitin nanofiber complex was prepared from crab shells by a simple mechanical treatment with a high-pressure water-jet (HPWJ) system. The preparation process did not involve chemical treatments, such as removal of protein and calcium carbonate with sodium hydroxide and hydrochloric acid, respectively. Thus, it was economically and environmentally friendly. The nanofibers obtained had uniform width and dispersed homogeneously in water. Nanofibers were characterized in morphology, transparency, and viscosity. Results indicated that the shell was mostly disintegrated into nanofibers at above five cycles of the HPWJ system. The chemical structure of the nanofiber was maintained even after extensive mechanical treatments. Subsequently, the nanofiber complex was found to improve the growth of tomatoes in a hydroponics system, suggesting the mechanical treatments efficiently released minerals into the system. The homogeneous dispersion of the nanofiber complex enabled easier application as a fertilizer compared to the crab shell flakes. Keywords: crab shell; chitin nanofiber; complex; fertilizer

1. Introduction Chitin is a highly abundant carbohydrate polymer occurring primarily in crab shells. Crab shells have a hierarchically-ordered organization [1]. The rigid chitin molecules are aligned in an antiparallel manner to form α-chitin nanofibers with an extended crystalline structure. These nanofibers are covered by a protein layer. The next layer consists of clusters of protein/chitin nanofibers that form a twisted plywood layer, which is gradually rotated about its normal axis. Calcium carbonate, consisting of calcite crystal, is embedded in the small cavity of the helicoidally-shaped structure. In a previous study, we isolated chitin nanofibers from crab shells and prepared them using a simple mechanical treatment [2]. Chitin nanofibers have a characteristic morphology [3], high surface-to-volume ratio [4], high mechanical strength [5,6], and efficient biological properties [7–10]. The chitin in crab shells is partially preserved in the fishing industry as an intermediate of chitosan and glucosamine, even though the other parts of the shell are disposed of as industrial waste. The chitin is prepared from crab shells by treatment with NaOH and HCl aqueous solutions to remove proteins and calcium carbonate [11]. A major effluent purification process is then required to purify the abundant calcium carbonate and protein residue, and the expense of this process is passed on in the cost of commercial chitin (currently about 5000 Japanese Yen/kg). In a previous study, we Int. J. Mol. Sci. 2016, 17, 1600; doi:10.3390/ijms17101600

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investigated methods for skipping the protein removal process to bring down production costs [12]. The protein/chitin nanofiber thus obtained, reinforce acrylic resin film[12]. and The increase methods for skipping the complex, protein removal process could to bring down production costs its mechanical properties. the protein the chitin nanofiber behaved as a substrate protein/chitin nanofiberMoreover, complex, thus obtained,layer couldonreinforce acrylic resin film and increase its mechanical properties. Moreover, protein layer thebasis chitinof nanofiber behaved as a substrate for for the biomineralization of calciumthe carbonate. Ononthe that study, we hypothesized that it of calcium carbonate. On shells the basis of that study, we hypothesized that it might the mightthebebiomineralization possible to convert the chitin in crab into nanofiber directly, without removing be possible to convert the chitinSimplification in crab shells into directly, process without removing the protein and protein and calcium carbonate. of nanofiber the preparation would significantly reduce calcium carbonate. Simplification of the preparation process would significantly reduce the production the production cost of nanofiber. Furthermore, eliminating the use of chemicals for purification would cost of nanofiber. Furthermore, eliminating the use of chemicals for purification would render the render the process more environmentally friendly. In this study, we report on the direct disintegration process more environmentally friendly. In this study, we report on the direct disintegration of crab of crab shells using our proposed method. We then characterize the obtained protein/CaCO3 /chitin shells using our proposed method. We then characterize the obtained protein/CaCO3/chitin nanofiber nanofiber complex inand detail, andits discuss itsapplication potential application as a plant since it is well complex in detail, discuss potential as a plant fertilizer since fertilizer it is well known that known that crab shells show enhancement of plant growth. crab shells show enhancement of plant growth. 2. Results andand Discussion 2. Results Discussion 2.1. Preparation andand Characterization /ChitinNanofiber Nanofiber Complex 2.1. Preparation Characterizationofofthe theProtein/CaCO Protein/CaCO33/Chitin Complex snow crab shellswere were used used as material in this The contents of the main Red Red snow crab shells asa astarting starting material in study. this study. The contents of the components in the crab shells (chitin, protein, and calcium carbonate) were estimated by the main components in the crab shells (chitin, protein, and calcium carbonate) were estimated by the ninhydrin-hydrindantin protein gravimetric analysis;they theywere wereapproximately approximately 30%, ninhydrin-hydrindantin protein testtest andand gravimetric analysis; 30%, 16%, 16%, and and 55% as w/w, respectively, indicating the largest proportion of the red crab shells was calcium 55% as w/w, respectively, indicating the largest proportion of the red crab shells was calcium carbonate. carbonate. Figure 1 shows field emission scanning electron microscope (FE-SEM) images of a crab shell Figure 1 shows field emission scanning electron microscope (FE-SEM) images of a crab shell after after high-pressure high-pressure water-jet (HPWJ) treatments with 0, 1, 5, 10, 30, and 50 passes. Before HPWJ water-jet (HPWJ) treatments with 0, 1, 5, 10, 30, and 50 passes. Before HPWJ treatment treatment (Figure 1a), the craborganization shell organization of protein/chitin nanofiber (Figure 1a), the crab shell consistedconsisted of protein/chitin nanofiber bundles withbundles calcium with calcium carbonate. one-pass treatment the organization had partially disintegrated, carbonate. AfterAfter one-pass treatment (Figure(Figure 1b), the1b), organization had partially disintegrated, and and nano-sized fiberswere wereobserved observedtotosome someextent. extent. morphological changes in crab the crab nano-sized fibers TheThe morphological changes in the shell shell one-pass treatment weredue due to to the loadload of the treatment. When the after after one-pass treatment were the strong strongmechanical mechanical ofHPWJ the HPWJ treatment. When number of passes increased from 5 to 50, the fiber’s width decreased further due to fibrillation of the number of passes increased from 5 to 50, the fiber’s width decreased further due to fibrillation protein/chitin nanofiber nanofiber bundle. grains alsoalso disintegrated into into smaller of thetheprotein/chitin bundle.Calcium Calciumcarbonate carbonate grains disintegrated smaller nanoparticles. Thus, the protein/CaCO3/chitin nanofiber complex was successfully obtained from nanoparticles. Thus, the protein/CaCO3 /chitin nanofiber complex was successfully obtained from crab shells by HPWJ mechanical treatments. To further characterize the morphology of the nanofiber, crab shells by HPWJ mechanical treatments. To further characterize the morphology of the nanofiber, calcium carbonate was removed from samples treated with 0, 1, and 5 passes. Calcium carbonate was calcium carbonate frominsamples 0, 1, and 5 passes. carbonate neutralized by was HCl removed and dissolved water astreated CaCl2. with SEM images revealed thatCalcium nanofibrillation of was neutralized HCl proceeded and dissolved in waterHPWJ as CaCl revealed that nanofibrillation 2 . SEM images the crabbyshells by repeated treatments and appeared complete at 5 passes (seeof the crab shells proceeded by repeated HPWJ treatments and appeared complete at 5 passes (see Figure S1). Figure S1).

Figure 1. Cont.

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Figure 1. FE-SEM micrographs of crab shells after (a) 0; (b) 1; (c) 5; (d) 10; (e) 30; and (f) 50 passes Figure 1. FE-SEM micrographs of crab shells after (a) 0; (b) 5; (d) 50 passes passes Figure 1. FE-SEM micrographs shells 0; length (b) 1; 1; (c) (c) (d) 10; 10; (e) (e) 30; 30; and and (f) (f) 50 through the high pressure water of jetcrab system. Theafter scale(a)bar is 15;µm. through the high pressure water water jet jet system. system. The scale bar length is is 11 µm. µm.

Figure 2 shows the FTIR-ATR spectra of pure chitin, calcium carbonate, and crab shells treated Figure 2 shows the FTIR-ATR spectra of pure chitin, calcium carbonate, and crab shells treated Figure 2 shows FTIR-ATR spectra of pure chitin, All calcium carbonate, and crab shells treated by by the HPWJ systemthe after 0, 1, 5, 10, 30, and 50 passes. spectra of the mechanically-treated crab by the HPWJ system after 0, 1, 5, 10, 30, and 50 passes. All spectra of the mechanically-treated crab the HPWJ after 0, 1, 5, 10, 30, andeach 50 passes. All spectra ofthat the mechanically-treated shells shells weresystem in excellent agreement with other. This suggests the original chemical crab structures shells were in excellent agreement with each other. This suggests that the original chemical structures were excellent agreement with each suggeststreatments. that the original chemical structures of the of thein crab shells were maintained afterother. HPWJThis mechanical In particular, the OH stretching of the crab shells were maintained after HPWJ mechanical treatments. In particular, the OH stretching −1, NH stretching −1, amide I crab shells were after HPWJ treatments. Inband particular, theand OH 1621 stretching band at 3424 cmmaintained bandmechanical at 3259 cm at 1652 cm−1,band and −1, and band at 3424 cm−1, NH stretching band at 3259 cm−1, amide I band at 1652 and−1621 −1 , NH −1 , amide 1 , andcm −1 at 3424 cm stretching band at 3259 cm I band at 1652 and 1621 cm amide II amide II band at 1554 cm of the chitin nanofibers were observed [11]. These absorption peaks amide II band at 1554 cm−1 of the chitin nanofibers were observed [11]. These absorption peaks − 1 −1 band at 1554 cm of the chitin nanofibers were observedbands [11]. These absorption peaks are 1450 especially are especially characteristic of chitin. The transmittance at approximately 870 and cm are especially characteristic of chitin. The transmittance bands at approximately 870 1450 cm−1 −1and characteristic chitin. The transmittance bandsOn at approximately 870the andpeaks 1450 cm were derived were derived offrom calcium carbonate [13]. the other hand, corresponding to were derived from calcium carbonate [13]. On the other hand, the peaks corresponding to from calcium carbonate [13].due On to thethe other hand, the of peaks corresponding to protein were notchitin observed, protein were not observed, weak nature the protein bands compared to their and protein were not observed, due to the weak nature of the protein bands compared to their chitin and due to3 the weak nature of the protein bands compared to their chitin and CaCO3 counterparts. CaCO counterparts. CaCO3 counterparts.

Figure spectra of of pure purechitin, chitin,calcium calciumcarbonate, carbonate,and andcrab crab shells treated with 0, 5, 1, 10, 5, 10, Figure 2. 2. FTIR-ATR FTIR-ATR spectra shells treated with 0, 1, 30, Figure 2. FTIR-ATR spectra of pure chitin, calcium carbonate, and crab shells treated with 0, 1, 5, 10, 30, and 50 passes through a high pressure water jet system. and 50 passes through a high pressure water jet system. 30, and 50 passes through a high pressure water jet system.

Figure 3 shows the light transmittances of a 0.1 wt % concentration, mechanically-treated crab shell showsthe thelight lighttransmittances transmittances a 0.1 % concentration, mechanically-treated crab Figure 33shows of of a 0.1 wt wt % concentration, mechanically-treated crab shell at 800 nm of water dispersion. Transmittance values were highly reproducible. Transparency is strongly shell at 800 nm of water dispersion. Transmittance values were highly reproducible. Transparency at 800 nm of water dispersion. Transmittance values were highly reproducible. Transparency is strongly associated with chitin fiber thickness because, when the solid fibers are dispersed at the nano level, the is stronglywith associated withthickness chitin fiber thickness because, when fibers at are dispersed at the associated chitin fiber because, when the solid fibersthe aresolid dispersed the nano level, suspension becomes transparent [14]. After HPWJ treatment, the crab shells remained well-dispersed nano level, the suspension becomes [14]. After HPWJ treatment, the crab shells remained suspension becomes transparent [14].transparent After HPWJ treatment, the crab shells remained well-dispersed in water for at least one month. Thus, the crab shells were easy to handle and shape into the desired well-dispersed in water for at least onethe month. Thus,were the crab wereand easyshape to handle anddesired shape in water for at least one month. Thus, crab shells easyshells to handle into the forms. Without HPWJ treatment, the light transmittance of crab shells was only 9.6%. As the number of into theWithout desiredHPWJ forms.treatment, Without HPWJ treatment, the light transmittance crab shells only 9.6%. forms. the light transmittance of crab shells was of only 9.6%. Aswas the number of passes increased, the thickness of the nanofibers decreased; and consequently, the light transmittance As the increased, number ofthe passes increased, thicknessdecreased; of the nanofibers decreased;the andlight consequently, the passes thickness of thethe nanofibers and consequently, transmittance of the dispersion increased. At 1 and 5 passes, the transmittance increased steeply to 12.7% and 17.1%, light of the dispersion At 1transmittance and 5 passes,increased the transmittance increased steeply to of thetransmittance dispersion increased. At 1 andincreased. 5 passes, the steeply to 12.7% and 17.1%, respectively. This shows that the composite nanofibers revealed high morphological change up to 12.7% and 17.1%, shows thatnanofibers the composite nanofibers revealed high morphological respectively. Thisrespectively. shows that This the composite revealed high morphological change up to 5 passes. Above 10 passes, the transparency did not change significantly, reaching only 25.5% at upAbove to 5 passes. Above 10transparency passes, the transparency did not change significantly, reaching only 5change passes. 10 passes, the did not change significantly, reaching only 25.5% at 50 passes. This trend indicates that—after up to five mechanical treatments—fibers were fibrillated 25.5% at 50This passes. trend that—after indicates that—after to five mechanical treatments—fibers were 50 passes. trendThis indicates up to five up mechanical treatments—fibers were fibrillated into thinner nanofibers, thus increasing their transparency. However, above 5 passes, the nanofibers fibrillated into thinner nanofibers, thus increasing their transparency. However, above 5 passes, the into thinner nanofibers, thus increasing their transparency. However, above 5 passes, the nanofibers were mostly fully fibrillated and further disintegration was difficult—resulting in saturated nanofibers were mostly fully fibrillated and further disintegration was difficult—resulting in saturated were mostly fully fibrillated and further disintegration was difficult—resulting in saturated transparency. These trends agreed with the results of FE-SEM. transparency. These trends transparency. trends agreed agreed with with the the results results of of FE-SEM. FE-SEM.

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Figure 3. Regular transmittances protein/CaCO33/chitin dispersions at 800 nmnm as as a Figure 3. Regular lightlight transmittances of of protein/CaCO /chitinnanofiber nanofiber dispersions at 800 a function of the number of passes. function of the number of passes. Figure 3. Regular light transmittances of protein/CaCO3/chitin nanofiber dispersions at 800 nm as

Figure 4 shows the viscosities a function of the number of passes. of 3.6 wt % mechanically-treated crab shell dispersions as a Figure shows the viscosities 3.6viscosity wt % mechanically-treated crab shell dispersions as of a function function4 of the number of passes.ofThe values were highly reproducible. The viscosity the of theslurry number of passes. The viscosity values were highly reproducible. The viscosity of the Figure 4 shows thefrom viscosities of 3.6 wt (cP) % mechanically-treated shell dispersions as slurry a sharply increased 3380 centipoise at zero passes to 9633crab cP at 5 passes. However, sharply increased 3380 centipoise (cP) at zero to 9633 cP at 5 passes. However, above function of the from number of passes. The viscosity values were reproducible. The viscosity of the above 10 passes, the viscosity decreased constantly topasses 2860 cPhighly at 50 passes. The viscosity was strongly slurry the sharply increased from 3380 centipoise at zero passes tochitin 9633 The cP atviscosity 5 passes. affected byviscosity nanofiber morphology [15]. The thinner and nanofibers are, However, the more 10 passes, decreased constantly to(cP) 2860 cPshorter at 50 the passes. was strongly above passes, the viscosityentangled, decreased constantly toand 2860 cP at 50 passes. The nanofibers viscosity wasare, strongly frequently the fibers become which increases theshorter viscosity. The viscosity data indicate that,more affected by10 nanofiber morphology [15]. The thinner the chitin the affected byfibers nanofiber morphology The as thinner and shorter theincreased. chitinThe nanofibers are, data the more up to 5 the passes, the nanofibers became[15]. thinner the number of passes Above approximately frequently become entangled, which increases the viscosity. viscosity indicate frequently the fibers become entangled, which increases the viscosity. The decreased viscosity data indicate that, 5 passes, the nanofibrillation was mostly completed, and the fiber length as fibers started that, up to 5 passes, the nanofibers became thinner as the number of passes increased. Above upbreak; to 5 passes, thelowering nanofibers thinner ason thethe number of passes increased. Above approximately to thereby thebecame viscosity. Based SEM observations, transparency, and viscosity approximately 5 passes, the nanofibrillation was mostly completed, and the fiber length decreased as 5 passes, the nanofibrillation wasbemostly completed, theshell fiber length decreased fibers started measurement results, it could inferred that theand crab composite fibers as were properly fiberstostarted break;lowering therebythe lowering the viscosity. Based on the SEM observations, break; to thereby viscosity. Based on the SEM observations, transparency, andtransparency, viscosity disintegrated and nanofibrillated at around 5 passes. and viscosity measurement results, could be inferred that thecomposite crab shellfibers composite fibers were measurement results, it could be itinferred that the crab shell were properly properly disintegrated and nanofibrillated at 5around disintegrated and nanofibrillated at around passes. 5 passes.

Figure 4. Viscosity of protein/CaCO3/chitin nanofiber dispersions as a function of the number of passes.

2.2. Figure Plant Effect the Protein/CaCO 3/Chitin Nanofiber Complex 4. Viscosity ofof protein/CaCO 3/chitin nanofiber dispersions as a function of the number of passes. Figure 4. Growth Viscosity of protein/CaCO 3 /chitin nanofiber dispersions as a function of the number of passes. Tomato plants grown in a hydroponic system were treated with nanofibers once a week. After 2.2. Plant Growth Effect of the Protein/CaCO3/Chitin Nanofiber Complex the fifth nanofiber treatment, the tomato plants treated with protein/CaCO3/chitin nanofibers exhibited 2.2. Plant Growth Effect the Protein/CaCO Nanofiber Complex Tomato plants grown in a diameter, hydroponic system were with nanofibers once week. markedly higher leafof age, stem and plant heighttreated than the controls (Figure 5, aTable 1).After The 3 /Chitin the fifth treatment, theon tomato plants treated with protein/CaCO 3/chitin exhibited effect of nanofiber the crab shell powder the tomato plant growth was comparable to nanofibers that of mineralized Tomato plants grown in astem hydroponic system were treated with nanofibers once a week. After the markedly higher leaf age, diameter, andinplant the controls (Figure 5, Table The chitin-protein composite nanofibers. As noted Tableheight 1, afterthan the fifth and ninth treatments, the 1). effects fifth nanofiber treatment, the tomato treated protein/CaCO nanofibers exhibited 3 /chitin effect the crab shellwere powder on plants the tomato plantwith growth wasfrom comparable that of mineralized of crabofshell powder not significantly different (p > 0.05) those oftoprotein/CaCO 3/chitin markedly higher leaf age, stem diameter, and plant height than the controls (Figure 5, Table 1). The chitin-protein composite Asofnoted inand Table 1, after the fifth andplants. ninth treatments, the effects effects nanofibers with respect tonanofibers. the number leaves stem diameter of the However, the effectof of the crab shell powder on the tomato plant growth was comparable to that of mineralized of the crab shell powder were notheight significantly differentafter (p >five 0.05) from those of significantly protein/CaCO 3/chitin two supplements on the of the tomatoes treatments were different nanofibers withthe respect to the number of leaves and stem diameter ofbetter the plants. effects chitin-protein nanofibers. As noted in Table 1, after the fifth and ninthHowever, treatments, the effects (p < 0.05);composite with protein/CaCO 3/chitin nanofibers exhibiting the effect on tomatothe growth. of shell the two supplements on height thedifferent tomatoes after treatments were The difference was attributed to theofeffect of mechanical treatment in producing the fastdifferent release of crab powder were notthe significantly (p > five 0.05) from those ofsignificantly protein/CaCO 3 /chitin (p < 0.05); with the protein/CaCO 3/chitin nanofibers exhibiting the better effect on tomato growth. of minerals deposited in the nanofibers network to the hydroponics system. In addition, nanofibers with respect to the number of leaves and stem diameter of the plants. However, the The difference was attributed effect of mechanical treatment in producing fast release mechanically-disintegrated 3/chitin nanofibers were easy totreatments apply due the to their liquid effects of the two supplementsprotein/CaCO onto thetheheight of the tomatoes after five were significantly of minerals deposited in the roughly-crushed nanofibers network to the hydroponics system. In addition, state, compared to the insoluble, crab shell powder. different (p < 0.05); with the protein/CaCO3 /chitin nanofibers exhibiting the better effect on tomato mechanically-disintegrated protein/CaCO3/chitin nanofibers were easy to apply due to their liquid growth. The difference was attributed to the effect of mechanical treatment in producing the fast state, compared to the insoluble, roughly-crushed crab shell powder.

release of minerals deposited in the nanofibers network to the hydroponics system. In addition, mechanically-disintegrated protein/CaCO3 /chitin nanofibers were easy to apply due to their liquid state, compared to the insoluble, roughly-crushed crab shell powder.

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Figure 5. Tomato plants grown hydroponically with nanofibers. Tomato plants were treated with

Figure 5. Tomato plants grown hydroponically with nanofibers. Tomato plants were treated with chitin chitin nanofiber, protein/chitin nanofiber, and protein/CaCO3/chitin nanofiber at a final concentration nanofiber, protein/chitin nanofiber, and protein/CaCO3 /chitin nanofiber at a final concentration of of chitin 0.01%. The series of nanofibers were prepared with high pressure water jet treatment with chitin0,0.01%. series of high pressure waterDistilled jet treatment with 0, 5, 5, and The 50 passes. Thenanofibers photo waswere takenprepared after the with fifth nanofiber treatment. water was and 50 passes. The photo was taken after the fifth nanofiber treatment. Distilled water was administered administered as a negative control and commercial nutrition (HYPONeX) as a positive control. as a negative controlcrab andshell commercial nutrition asscale a positive control. Roughly-crushed Roughly-crushed powder was treated (HYPONeX) as a control. The bar length is 36 mm. crab shell powder was treated as a control. The scale bar length is 36 mm. Table 1. The effect of protein/CaCO3/chitin nanofibers on the growth of tomato plants.

Table Day after 1. Treatment Day after Treatment

The effect of protein/CaCO nanofibers growth of tomato Plant plants. 3 /chitin Number of Number of on the Stem Diameter Height Treatment

Distilled water Treatment HYPONeX Crab shell

2.0 ± 0.1 c

(cm) 0.9Height ± 0.1 a Plant 11.6 ± 0.8 b (cm) 4.3 ± 0.2 c,d,e

1.4 ± 0.0 a 2.2 ± 0.1 c 3.0 ± 0.2 b 2.0 ± 0.1 c 2.0 ± 0.1 c

0.9 ± 0.1 a 6.8 ± 1.9 c 11.6 ± 0.8 b c,d 4.4 ± 0.4 4.3 ± 0.2 c,d,e

00 55 50 50

6.3 0.2 ac 3.1±± 0.2 5.8 ± 0.3 ac 3.5 ± 0.2 6.7 0.2 ac 3.6±± 0.2

c 2.21.4 ±±0.1 0.0 a c 2.01.5 ±±0.1 0.0 a c 2.21.5 ±±0.1 0.0 a

c 6.81.0 ± ±1.9 0.1 a c,d 4.41.4 ±±0.4 a,d,e 0.1 c 5.01.2 ±±0.1 0.1 a,e

Chitin Protein/chitin nanofiber nanofiber

00 55 50 50

3.2±± 0.1 3.1 0.2 aa 3.0±± 0.3 3.5 0.2 aa 3.6 0.2 aa 3.0±± 0.1

0.0 aa 1.41.4 ±±0.0 0.0 aa 1.51.4 ±±0.0 1.51.4 ±±0.0 0.0 aa

0.1aa 1.01.0 ± ±0.1 a,d,e a 1.00.1 ± 0.1 1.4 ± a,ea 1.2 0.9 ± 0.1 ± 0.0

Distilled water Chitin HYPONeX nanofiber Crab shell

-0 -5 50

3.2 ± 0.1 a a 9.7±± 0.4 3.0 0.3 a 8.4 ± 0.5 3.0 ± 0.1 aa

1.4 ±ND 0.0 a 0.1 aa 1.43.3 ±±0.0 2.1 ± 0.1 1.4 ± 0.0 ab

1.0 ± 0.1 a ± 0.5a a 1.016.6 ± 0.1 7.0 ± 0.9ab 0.9 ± 0.0

0 5 50

9.1 ± 0.3 a 8.6 ± 0.3 aa 9.7 ± 0.4 a 9.2 ± 0.3 a

2.2 ± 0.1 b ND 2.2 ± 0.1 b 3.3 ± 0.1 ab 2.3 ± 0.1b

Protein/CaCO Protein/chitin 3 / chitin nanofiber nanofiber

Protein/CaCO / Distilled water 3

HYPONeX chitin nanofiber Crab shell Protein/ Protein/CaCO chitin nanofiber 3 /

9 weeks

6.0 ± 0.3 c

(mm) ± 0.0 a Stem1.4 Diameter 3.0 ± 0.2 b (mm)

3.2 ± 0.1 a 6.3 ± 0.2 c 8.3 ± 0.2 b 5.8 ± 0.3 cc 6.0 ± 0.3

5 weeks

9 weeks

-

Leaves a 3.2 ± 0.1 of Number 8.3 ± 0.2 b Leaves

-

Distilled water HYPONeX3/ Protein/CaCO Crab shell chitin nanofiber

5 weeks

Passes Number of Passes

chitin nanofiber Chitin nanofiber Protein/chitin

0 5 50

-

0 50 505 050 50 505

6.7 ± 0.2 c

2.2 ± 0.1 c

5.0 ± 0.1 c

8.4 ± 0.5

2.1 ± 0.1

9.1 ± 0.6 b 7.8 ± 0.6 b 16.6 ± 0.5 ab 9.3 ± 0.5b

9.1 ± 0.3 a 8.6 ± 0.3 a 9.2 ± 0.3 a

2.2 ±ND 0.1 b 2.2 ±ND 0.1 b 2.3 ±ND 0.1 b

9.1 ± 0.6 b 7.8 ± 0.6 b 9.3 ± 0.5 b

ND

7.0 ± 0.9

ND ND ND ND nanofiber 50 ND with the same letter Data represents the mean of four independent experiments and error bars. Means

(a–e) are not significantly different according to Tukey’s test (p < 0.05) in ND each week. ND indicates 0 Chitin dead plant. 5 ND nanofiber 50 ND Data represents the mean of four independent experiments and error bars. Means with the same letter (a–e) are not significantly different according to Tukey’s test (p < 0.05) in each week. ND indicates dead plant.

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On the other hand, neither protein/chitin nanofibers nor chitin nanofibers had any effect on tomato growth. Tomato plants treated with protein/chitin nanofiber, chitin nanofiber and distilled water (DW) died at the ninth nanofibers treatment; whereas tomato plants treated with protein/CaCO3 /chitin nanofibers or crab shells showed healthy growth. These results indicated that the protein/CaCO3 /chitin nanofibers might act as a fertilizer. Plants require not only essential elements—such as nitrogen, phosphorus, and potassium—but also calcium, sulfur, magnesium, and trace minerals for their growth. Although chitin nanofibers and protein/chitin nanofibers contain essential nitrogen, these nanofibers might not contain sufficient amounts of minerals to promote plant growth. Even though the effects of protein/CaCO3 /chitin nanofibers were not as dramatic as those of the commercial nutrient HYPONeX; after further improvements, nanofibers might show good performance as a fertilizer. The results in Table 1 also revealed that there were no significant differences among the nanofibers treated with a different number of passes of the HPWJ system in terms of tomato growth. Therefore, the 0 pass sample of protein/CaCO3 /chitin nanofibers, which was roughly crushed by grinder treatment with two passes, might be a cost-effective material for use as a fertilizer for plant growth. 3. Materials and Methods 3.1. Materials The raw shells of Chionoecetes opilio (red snow crab) and α-chitin powder with a 6.4% deacetylation degree were obtained from Koyo Chemicals. Other chemicals were purchased from Aldrich or Kanto Chemicals and were used as received. Tomato seeds were purchased from the Marutane Seed Co., Ltd., Kyoto, Japan. 3.2. Preparation of the Protein/CaCO3 /Chitin Nanofiber Complex Raw crab shells were roughly crushed with a domestic blender under wet conditions. The crushed sample was then disintegrated with a grinder (MKCA6-3; Masuko Sanyo Co., Ltd., Kawaguchi, Japan) at 1500 rpm for two cycles. The air bubbles were removed by a super stirrer (Awatori-rentaro, ARE-301, THINKY, Ltd., Tokyo, Japan). The concentration of the total solid was adjusted to 3.6 wt % for a chitin weight percentage of around 1 wt %. The samples were then passed through a high-pressure water-jet system (HPWJ; Star Burst Mini, HJP-25001S, Sugino Machine, Namerikawa, Japan) equipped with a ball collision chamber. The slurry was ejected from a small nozzle with a diameter of 100 µm under high pressure of 200 MPa. This mechanical treatment was repeated for 1, 5, 10, 30, and 50 cycles. 3.3. Preparation of the Protein/Chitin Nanofiber Complex and Pure Chitin Nanofibers Protein/chitin nanofiber complex and pure chitin nanofibers were prepared in order to compare their effects on tomato growth with those of the protein/CaCO3 /chitin nanofiber complex. The protein/chitin nanofiber complex was prepared using the previously reported procedure, with slight modifications [12]. Briefly, the raw crab shells were crushed with a domestic blender under wet conditions. The crushed sample was treated with 2 M hydrochloric acid for two days at room temperature, with occasional stirring to remove mineral salts (mainly calcium carbonate). The sample was collected by filtration and washed with deionized water until the filtrate became neutral. The sample was then crushed by grinder treatment, and the concentration of the overall solid was adjusted to 1.15 wt %. The ground sample was passed through 5 and 50 cycles of a high-pressure water-jet (HPWJ) system. For comparison, pure chitin nanofiber was also prepared as described in our previous study, and then modified as follows [16]. Dry pure chitin powder was dispersed in water at 1 wt %. After 1 h of stirring, the dispersion was crushed with a grinder for two cycles. The ground sample was stirred under a vacuum for 1 h to remove air bubbles. Finally, it was mechanically homogenized with 5 and 50 cycles of the HPWJ system.

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3.4. Characterization For field emission scanning electron microscopic (FE-SEM) observation, the disintegrated samples were diluted with ethanol and dried in an oven. The dried samples were coated with an approximately 2 nm layers of platinum, using an ion sputter coater, and were observed by an FE-SEM (JSM-6700F; JEOL, Tokyo, Japan) operating at 2.0 kV. Infrared spectra of the samples was recorded with an FT-IR spectrophotometer (Spectrum 65; Perkin-Elmer Japan, Yokohama, Japan) equipped with an ATR attachment (diamond/ZnSe crystal) with four scans at a resolution of 4 cm−1 . The light transmittances of the protein/mineral/chitin nanofiber complex dispersion were measured using a UV-Vis spectrophotometer (V550; JASCO, Tokyo, Japan). The viscosities of the complex slurries were measured with a Brookfield digital viscometer DV-E using the LV-4 spindle (Brookfield Engineering Laboratories, Middleboro, MA, USA) at 30 ◦ C. The protein, calcium carbonate, and chitin contents of the original crab shells were estimated by means of a ninhydrin-hyrindantin protein test [17] and gravimetric analysis. Calcium carbonate and protein were separated from the crab shells by conventional 1 M HCl and 1 M KOH treatments, respectively. 3.5. Plant Material Tomato (Solanum lycopersicum) cv. Chibikko (Marutane Seed) was grown hydroponically with rockwool grow cubes (AO 36/40; Grodan). Three weeks after seeding, the tomato plants were treated with a series of nanofibers, which contained chitin at a final concentration of 0.01% to each rockwool cube. The number of true leaves, the stem diameter (at cotyledon node), and the plant height (stems length above the cotyledon) were measured after the fifth and ninth nanofiber treatment. The plants were grown under environmentally-controlled conditions with 14 h of light/10 h of dark cycles at 25 ◦ C, and treated with nanofibers once a week. Distilled water (DW) was used as a negative control, and commercial nutrition HYPONeX (N-P-K = 6-10-5; Hyponex Japan, Osaka, Japan) at a concentration of 0.001% was used as a positive control. Roughly-crushed crab shell powder was treated as a control. 4. Conclusions A protein/CaCO3 /chitin nanofiber complex was prepared from crab shells using a simple mechanical treatment. Because this nanofiber preparation process did not include the conventional protein and calcium carbonate removal processes, it could bring down the production cost and environmental load. The resulting protein/CaCO3 /chitin nanofiber would be available as a fertilizer to improve plant growth in hydroponic systems. Indeed, the mineral part of the nanofiber complex was found to be vital for plant growth and maturation. Finally, the novel nanofiber complex could be prepared at a low cost through an eco-friendly process. Thus, the results of this close analysis of the unique nanofiber-complex characteristics could lead to more effective utilization of crab shell waste. Supplementary Materials: Supplementary materials can be found at www.mdpi.com/1422-0067/17/10/1600/s1. Acknowledgments: This research was supported by Grant-in-Aid for Scientific Research (Young Scientists (A), No. 26708026), Japan Society for the Promotion of Science, and by Science and Technology Research Promotion Program for Agriculture, Forestry, Fisheries and Food Industry. Author Contributions: Shinsuke Ifuku and Hironori Kaminaka conceived and designed the experiments; Yihun Fantahun Aklog and Mayumi Egusa performed the experiments; Yihun Fantahun Aklog, Mayumi Egusa, Minoru Morimoto, Hiroyuki Saimoto and Hironori Izawa analyzed the data; Yihun Fantahun Aklog, Mayumi Egusa and Shinsuke Ifuku wrote the paper. Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of the data; in the writing of the manuscript, and in the decision to publish the results.

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