marine drugs Article
Microbial Degradation Behavior in Seawater of Polyester Blends Containing Poly(3-hydroxybutyrateco-3-hydroxyhexanoate) (PHBHHx) Hitoshi Sashiwa 1, *, Ryuji Fukuda 1 , Tetsuo Okura 2 , Shunsuke Sato 3 and Atsuyoshi Nakayama 4 1 2 3 4
*
BDP Group, New Buisiness Development Department, Kaneka Co., 5-1-1, Torikai-Nishi, Settsu, Osaka 566-0072, Japan;
[email protected] BDP Processing Technology Development Team, Plastics Molding & Processing Technology Development Group, Kaneka Co., 5-1-1, Torikai-Nishi, Settsu, Osaka 566-0072, Japan;
[email protected] BDP Group, Biotechnology Development Laboratories, Kaneka Co., 8-1 Miyamae-cho, Takasago, Hyogo 676-8688, Japan;
[email protected] Biomolecule Design Research Group, Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-8-31, Midorigaoka, Ikeda, Osaka 563-8577, Japan;
[email protected] Correspondence:
[email protected]; Tel.: +81-72-653-8335
Received: 29 November 2017; Accepted: 15 January 2018; Published: 17 January 2018
Abstract: The microbial degradation behavior of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) and its compound with several polyesters such as poly(butylene adipate-co-telephtharate) (PBAT), poly(butylene succinate) (PBS), and polylactic acid (PLA) in seawater was tested by a biological oxygen demand (BOD) method. PHBHHx showed excellent biodegradation in seawater in this study. In addition, the biodegradation rate of several blends was much influenced by the weight ratio of PHBHHx in their blends and decreased in accordance with the decrement of PHBHHX ratio. The surface morphology of the sheet was important factor for controlling the biodegradation rate of PHBHHx-containing blends in seawater. Keywords: poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx); blendsurface morphology; microbial; degradation; seawater
1. Introduction In recent years, there is increasing interest in marine pollution owing to the effect of microplastics [1,2]. Microplastics are defined as particles of less than 5 mm in size and depending on the plastic, it has been shown that various chemicals and toxic blends can be absorbed onto their surface [3]. For this, it is great concern that these blends could be accumulated into zooplankton, fish, and finally to mammals through the food chain. Therefore, the dynamics of production, distribution, diffusion, and degradation of microplastics in marine environments are receiving a lot of attention. One of the possible solutions toward this problem is focused on the application of biodegradable plastics. Polyhydroxyalkanoates (PHAs) have the most promising biodegradable properties amongst the class of several biodegradable plastics [4,5]. Since PHA plastics degrade and disappear in seawater, toxic blends bound microplastics have no existence in marine environment [6]. Among the PHAs, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx, Figure 1) shows excellent flexibility, because the 3-hydroxyhexanoate (3-HHx) unit (Figure 1b) is not taken into the crystallized 3-hydroxybutyrate (3-HB) unit (Figure 1a) in the crystal structure. Therefore, the crystallinity of this polymer can be controlled by changing the percentage of the 3-HHx unit [7].
Mar. Drugs 2018, 16, 34; doi:10.3390/md16010034
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(a)
(b)
Figure 1. Chemical structure of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx). Figure 1. Chemical structure of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx). (a) hydroxybutyrate hydroxybutyrate (3-HB) (3-HB) unit; unit; (b) (b) 3-hydroxyhexanoate 3-hydroxyhexanoate (3-HHx) (3-HHx) unit. unit. (a)
PHAs including PHBHHx show good biodegradation properties in soil, as for other PHAs including PHBHHx good biodegradation properties in soil, as (PBAT), for other biodegradable biodegradable polyesters suchshow as poly(butylene adipate-co-telephtharate) poly(butylene polyesters such as poly(butylene adipate-co-telephtharate) (PBAT), poly(butylene succinate) (PBS), succinate) (PBS), poly(butylene succinate-co-adipate) (PBSA), polycaprolactone (PCL), etc. [8–10]. poly(butylene succinate-co-adipate) (PBSA), (PCL), etc. [8–10]. Especially, it has been Especially, it has been reported that PHAspolycaprolactone such as PHB and poly(3-hydroxybutyrate-co-valerate) reported that PHAs such as PHB and poly(3-hydroxybutyrate-co-valerate) PCL showed (PHBV) or PCL showed excellent biodegradation in seawater [11–16]. By (PHBV) contrast,orpolylactic acid excellent biodegradation in seawater [11–16]. By contrast, polylactic acid (PLA), which is one under of the (PLA), which is one of the most utilized biodegradable polyesters, shows poor degradability most utilized biodegradable polyesters, shows degradability under ambient conditions, although ambient conditions, although it is possible to poor degrade under industrial composting conditions [17]. it is possible to degrade under industrial composting conditions [17]. On the other hand, PHAs are On the other hand, PHAs are polyesters biologically synthesized by various microorganisms from polyesters biologically synthesized by various microorganisms from renewable resources such as renewable resources such as saccharides, vegetable oils, glycerol, and others. PHA-degrading saccharides, oils, glycerol, and environments others. PHA-degrading bacteria have (extracellular been isolated PHA from bacteria havevegetable been isolated from most and produce enzymes most environments and produce enzymes (extracellular PHA depolymerase) to degrade PHAs under depolymerase) to degrade PHAs under both anaerobic and aerobic conditions. In general, PHAs both anaerobic andshowed, aerobic conditions. general, PHAs include showed, blends with other include PHBHHx blends withInother polyesters such asPHBHHx PBAT, PLA, and PBS were carried polyesters such as PBAT, PLA, and PBS were carried out to make up the mechanical properties [10]. out to make up the mechanical properties [10]. Therefore, the biodegradability of these blends in Therefore, the biodegradability of these blends in seawater needs to be investigated. seawater needs to be investigated. In of different In this this study, study, we we investigated investigated the the effect effect on on seawater seawater biodegradability biodegradability of different highly highly biodegradable (as PBAT and PBS) and poorly biodegradable (as PLA) polyesters in the biodegradable (as PBAT and PBS) and poorly biodegradable (as PLA) polyestersblends in the(powder blends and sheets) PHBHHx at different ratios. ratios. Because PHBHHx showsshows excellent flexibility and (powder andwith sheets) with PHBHHx at different Because PHBHHx excellent flexibility heat resistant properties, PHBHHx is used in notinonly its independent form form but also blends with and heat resistant properties, PHBHHx is used not only its independent but as also as blends several biodegradable polyesters. Moreover, the effect of surface morphology of PHBHHx/PBAT with several biodegradable polyesters. Moreover, the effect of surface morphology and of PHBHHx/PLA sheets was also investigated in this study. PHBHHx/PBAT and PHBHHx/PLA sheets was also investigated in this study. 2. Results and Discussion 2. Results and Discussion 2.1. Biodegradation of PHBHHx and PHBHHx/PBAT Blends 2.1. Biodegradation of PHBHHx and PHBHHx/PBAT Blends In this study, 11 mol% of 3-HHx unit content of PHBHHx was used. Table 1 shows the In this study, 11 mol% of the 3-HHx unit content of of PHBHHx was used. Table 1 shows theand bending bending elastic modulus and molecular weight polymers used in this study before after elastic modulus and the molecular weight of polymers used in this study before and after biodegradation in seawater for 28 days. PBAT, PBS, and PLA, which were not shown microbial biodegradation in seawater for 28 days. PBAT, PBS, and PLA, which were not shown microbial metabolism estimated by biological oxygen demand (BOD) test, showed slight decrease of molecular metabolism estimatedinby biological demand test,which showed slight microbial decrease of molecular weight by hydrolysis seawater. Onoxygen the other hand, (BOD) PHBHHx, showed metabolism, weight by hydrolysis seawater. OnThis thephenomenon other hand,could PHBHHx, which as showed did not show molecular in weight change. be explained follows:microbial first the metabolism, did not show molecular weight change. This phenomenon could be explained as follows: surface of PHBHHx was hydrolyzed by microbial enzymes, and then seawater soluble oligomers of first the surface of PHBHHx was hydrolyzed by microbial enzymes, and then seawater soluble PHBHHx were metabolized by microorganisms in seawater. Since it has been shown that PHBHHx oligomers of resistance PHBHHx of were metabolized by microorganisms seawater. it hasthe been shown showed high hydrolysis in comparison with those in of PBAT, PBS,Since and PLA, remained that PHBHHx showed high resistance of hydrolysis in comparison with those of PBAT, PBS, and PBHHx would still keep the high molecular weight. PLA, the remained PBHHx would still keep the high molecular weight.
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Table 1. The molecular weight of polymers (sheet shape) used in this study before and after Mar. Drugs 2018, 16, 34 3 of 11 biodegradation in seawater for 28 days. Polymer
Table 1. The molecular weight of polymers (sheet shape) used in this study before and after Bending Elastic Modulus After 28 Days Biodegradation biodegradation in seawater for 28 days. Before Biodegradation Polymer
PHBHHx PBAT PHBHHx PBS PBAT PLA PBS PLA
MPa M Mw Mw/M M Mw Mw/Mn Bending Elastic Modulus n Before Biodegradation n After 28 nDays Biodegradation 850 280,000 Mn 550,000 2.0 n 1.9 MPa Mw Mw/M M280,000 n Mw 530,000 Mw/Mn 110 63,000280,000120,000 1.9 55,000 530,000 110,0001.9 2.0 850 550,000 2.0 280,000 650 81,00063,000 150,000 1.9 77,000 110,000 126,0002.0 1.6 110 120,000 1.9 55,000 3190 100,000 160,000 1.6 94,000 140,000 1.5 650 81,000 150,000 1.9 77,000 126,000 1.6 3190 100,000 160,000 1.6 94,000 140,000 1.5
Figure 2 shows the typical time courses of the biodegradation of PHBHHx and PHBHHx/PBAT Figure 2 shows the typical time courses of the biodegradation of PHBHHx and PHBHHx/PBAT blends (powder form) in seawater as indicated by their biological oxygen demand (BOD). blends (powder form) in seawater as indicated by their biological oxygen demand (BOD). The The biodegradation increased with degradation ThePHBHHx pure PHBHHx biodegradationofofPHBHHx PHBHHx gradually gradually increased with the the degradation time. time. The pure showedshowed excellent biodegradability in seawater in comparison with the original excellent biodegradability in seawater in comparison with the original PBAT,PBAT, whose whose biodegradability level.The The degree degree of therefore showed much much biodegradability was was quitequite lowlow level. of biodegradation biodegradation therefore showed dependence the effect of the weight ratioininthe the blend. blend. The biodegradation was decreased with thewith the dependence on theon effect of the weight ratio The biodegradation was decreased of PHBHHx. ratio of ratio PHBHHx.
Figure The biodegradation of PHBHHxand andPHBHHx/PBAT PHBHHx/PBAT blends (powder shape)shape) in seawater. Figure 2. The 2. biodegradation of PHBHHx blends (powder in seawater. ●: PHBHHx, ○: PHBHHx/PBAT = 80/20 (wt/wt), ▲: PHBHHx/PBAT = 60/40 (wt/wt), : PHBHHx, #: PHBHHx/PBAT = 80/20 (wt/wt), N: PHBHHx/PBAT = 60/40 (wt/wt), ■: PHBHHx/PBAT = 40/60 (wt/wt), ♦: PBAT. : PHBHHx/PBAT = 40/60 (wt/wt), : PBAT.
The biodegradation degree (%) in seawater after 28 days is summarized in Table 2. The particle distribution of all powder(%) form samples were 24–2000 μm. Thesize biodegradation degree inof seawater afteraround 28 days is summarized in Table 2. The particle To confirm only the PHBHHx component degrade in the blends, the content (wt %) of PHBHHx size distribution of all powder form of samples were around 24–2000 µm. in the blends were analyzed by 1H-NMR analysis before and after biodegradation (Figure S1). The To confirm only the PHBHHx component degrade in the blends, the content (wt %) of PHBHHx contents of PHBHHx before biodegradation were a little bit changed after the melt exclusion process. in the blends were analyzed by 1 H-NMR analysis beforedecreased and after biodegradation (Figure S1). As shown in Table 2, the contents of PHBHHx were slightly after biodegradation, which The contents of PHBHHx before biodegradation were a little bit changed after the melt exclusion means that PHBHHx component surely degraded with seawater. process. As shown in Table 2, the contents of PHBHHx were slightly decreased after biodegradation, Table 2. The biodegradation degree (%) ofdegraded PHBHHx/PBAT in seawater after 28 days. which means that PHBHHx component surely withblends seawater. PHBHHx/PBAT
Content of PHBHHx
Average Particle
in Blends (wt %) 28 days. Form (%) of PHBHHx/PBAT Biodegradation (%)in seawater Table 2.Entry The biodegradation degree blends after Added (wt/wt) Size (μm)
Entry 1 2 3 4 5
1 100/0 2 80/20 PHBHHx/PBAT 3 60/40 Added (wt/wt)40/60 4 5 0/100
100/0 80/20 60/40 40/60 0/100
Powder Powder FormPowder Powder Powder
Powder Powder Powder Powder Powder
440 380 Average Particle 400 Size (µm) 400 470 (a)
(a)
440 tested. Not 380 400 400 470 Not tested.
Before 31 - (a) 19 83 Biodegradation (%) 10 66 3 47 1 -
31 19 10 3 1
After Content of PHBHHx 80 in61Blends (wt %) 46 Before After -
- (a) 83 66 47 -
80 61 46 -
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2.2. Biodegradation of PHBHHx/PBS Blends 2.2. Biodegradation of PHBHHx/PBS Blends The biodegradation degree (%) of PHBHHx/PBS blends in seawater after 28 days is summarized The biodegradation degree (%) of PHBHHx/PBS blends in seawater after 28 days is summarized in Table 3. In the case of PHBHHx/PBS, the biodegradability also showed much dependence on the in Table 3. In the case of PHBHHx/PBS, the biodegradability also showed much dependence on the PBS ratio in the blends. On comparing with the relative rates (calcd. and found), PHBHHx/PBS = PBS ratio in the blends. On comparing with the relative rates (calcd. and found), PHBHHx/PBS = 80/20 (entry 2) was not found to inhibit the biodegradation rate. However, the 60/40 and 40/60 blends 80/20 (entry 2) was not found to inhibit the biodegradation rate. However, the 60/40 and 40/60 blends (entries 3 and 4) showed the same level of inhibition as the PBAT blends. These results suggest (entries 3 and 4) showed the same level of inhibition as the PBAT blends. These results suggest that that a high amount of PBS also inhibits the biodegradability of PHBHHx. In these cases, PHBHHx a high amount of PBS also inhibits the biodegradability of PHBHHx. In these cases, PHBHHx components were surely decreased after biodegradation. The content (wt %) of PHBHHx in the blends components were surely decreased after biodegradation. The content (wt %) of PHBHHx in the were analyzed by 1 H-NMR analysis before and after biodegradation (Figure S3). And Figure S4 shows blends were analyzed by 1H-NMR analysis before and after biodegradation (Figure S3). And Figure the time courses of the biodegradation of PHBHHx/PLA blends (powder form) in seawater. S4 shows the time courses of the biodegradation of PHBHHx/PLA blends (powder form) in seawater. Table 3. The effect of mixture ratio of PHBHHx/PBS blends on the biodegradation in seawater after 28 days. Table 3. The effect of mixture ratio of PHBHHx/PBS blends on the biodegradation in seawater after 28 days. PHBHHx/PBS Entry Entry PHBHHx/PBS Form Form (wt/wt) (wt/wt) 1 2 3 4 5
1 2 3 4 5
100/0 100/0 80/20 80/20 60/40 60/40 40/60 40/60 0/100 0/100
Average AverageParticle Particle Size Size (µm) (μm)
Powder Powder Powder Powder Powder Powder Powder Powder Powder Powder
440 440 370 370 420 420 390 390 310 310 (a) (a)
Content of PHBHHx Content of PHBHHx Biodegradation Biodegradation (%) in Blends in Blends (wt %) (wt %) (%) Before After After Before 51 51 - (a) - (a) 41 41 80 80 76 76 18 18 61 61 57 57 5 5 42 42 38 38 1 1 -
Not tested. Not tested.
2.3. Biodegradation of PHBHHx/PLA Blends Figure 3 shows the time courses of the biodegradation biodegradation of of PHBHHx/PLA PHBHHx/PLA blends blends (powder (powder form) form) in seawater. showed quite lowlow biodegradability in seawater, as for and seawater. The Theoriginal originalPLA PLA showed quite biodegradability in seawater, as the for PBAT the PBAT PBS. However the biodegradation of PHBHHx/PLA blends showed less dependence on theonPLA and PBS. However the biodegradation of PHBHHx/PLA blends showed less dependence the ratio. The biodegradation degree degree (%) in seawater after 28 after days 28 is summarized in Table 4. these PLA ratio. The biodegradation (%) in seawater days is summarized in In Table 4. cases, PHBHHx components were surely decreased after biodegradation. The reasonThe of relatively In these cases, PHBHHx components were surely decreased after biodegradation. reason of high biodegradation the PHBHHx/PLA blendsblends of 80/20, 60/40, andand 40/60 is isdiscussed relatively high biodegradation the PHBHHx/PLA of 80/20, 60/40, 40/60 discussed later later 1 1 (Section 2.5). The content (wt %) of PHBHHx in the blends were analyzed by H-NMR H-NMR analysis analysis before before and after biodegradation biodegradation(Figures (FiguresS2-1 S2-1and andS2-2). S2-2).And AndFigure FigureS5S5shows shows the particle size distribution the particle size distribution of of PHBHHx/PLA blend powders. PHBHHx/PLA blend powders.
Figure 3. The biodegradation of PHBHHx and and PHBHHx/PLA PHBHHx/PLA blends Figure 3. The biodegradation of PHBHHx blends(powder (powdershape) shape)in in seawater. seawater. ●:: PHBHHx, ○: PHBHHx/PLA == 80/20 = PHBHHx, #: 80/20(wt/wt), (wt/wt),▲: N: PHBHHx/PLA PHBHHx/PLA==60/40 60/40(wt/wt), (wt/wt),■:PHBHHx/PLA : PHBHHx/PLA 40/60 (wt/wt), ♦: PLA. = 40/60 (wt/wt), : PLA.
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Table 4. The effect of mixture ratio of PHBHHx/PLA blends on the biodegradation in seawater after 28 days.
Table 4. The effect of mixture ratio of PHBHHx/PLA blends on the biodegradation in seawater after 28 days. Entry 1 2 3 4 5
PHBHHx/PLA Form PHBHHx/PLA Entry (wt/wt) Form (wt/wt) 1 2 3 4 5
100/0 80/20 60/40 40/60 0/100
100/0 80/20 60/40 40/60 0/100
Powder Powder Powder Powder Powder Powder Powder Powder Powder Powder
Average Particle Biodegradation (%) Average Particle Biodegradation Size (µm) Size (μm) (%) 440 440 420 420 430 430 440 440 490 490 (a) Not tested. (a)
34 34 33 33 32 32 26 126 1
Content of PHBHHx Content of PHBHHx in Blends (wt %) in Blends (wt %) After Before Before After - (a) - (a) 82 77 82 77 64 62 64 62 51 45 5145 -
Not tested.
2.4. Effect of 2.4. Effect of the the Morphology Morphology of of PHBHHx/PBAT PHBHHx/PBAT Compound Compound on on the the Biodegradation Biodegradation Table Table 55shows showsthe theeffect effectof ofthe theform formof ofthe thePHBHHx/PBAT PHBHHx/PBAT blends blends on on the the biodegradation biodegradation rate rate in in seawater after 28 days. In the most cases, the sheet forms (entries 2 and 4) showed less biodegradability seawater after 28 days. In the most cases, the sheet forms (entries 2 and 4) showed less in comparison with those of powder form.ofTo clarifyform. this reason for this these difference, transmission biodegradability in comparison with those powder To clarify reason for these difference, electron microscope (TEM) and scanning electron microscope (SEM) microscope analyses were performed. Figure 4 transmission electron microscope (TEM) and scanning electron (SEM) analyses were shows the SEM image of the RuO4 stained PHBHHx/PBAT = 80/20 and 60/40 blend sheets surface performed. Figure 4 shows the SEM image of the RuO4 stained PHBHHx/PBAT = 80/20 and 60/40 before biodegradation. The PBAT was the brighter part owing to a brighter higher scattering intensity of the blend sheets surface before biodegradation. The PBAT was the part owing to a higher electron beam by the attachment of RuO to the phenyl group in PBAT compared with the PHBHHx 4 scattering intensity of the electron beam by the attachment of RuO4 to the phenyl group in PBAT component. Despite the highcomponent. PHBHHx fraction = 80/20inand 60/40, a large= compared with the PHBHHx Despite in thePHBHHx/PBAT high PHBHHx fraction PHBHHx/PBAT proportion of the PBAT component was at component the surface of these sheets, which 80/20 and 60/40, a large proportion ofobserved the PBAT was observed at the would surfacebeofcaused these by the lower molecular weight of PBAT layer tends to exposure at the surface area under preparing sheets, which would be caused by the lower molecular weight of PBAT layer tends to exposure at the T-die sheet. would explainT-die the decrease of biodegradation for the PBAT of blends because thefor PBAT surface areaThis under preparing sheet. This would explain the decrease biodegradation the at the surface restrictedthe microorganial PHBHHx. PBAT blends because PBAT at the access surfacetorestricted microorganial access to PHBHHx. Figure shows the theTEM TEMphotograph photographofofa cross a cross section of the PHBHHx/PBAT = 80/20 and Figure 55 shows section of the PHBHHx/PBAT = 80/20 and 60/40 60/40 biodegradation. Thecomponent PBAT component wasowing darkstoowing to less permeability of sheets sheets before before biodegradation. The PBAT was darks less permeability of electron electron beam through the attachment of RuO to the phenyl group in the PBAT compared with the 4 beam through the attachment of RuO4 to the phenyl group in the PBAT compared with the PHBHHx PHBHHx PBAT layer structure (blackwas parts) was arranged an annual the part. The part. PBATThe layer structure (black parts) arranged like an like annual ring ofring treeof fortree thefor 60/40 60/40 in comparison of 80/20 which more irregular. Thisdifference differencemeans means that that the blend blend in comparison with with that that of 80/20 which waswas more irregular. This the biodegradation was more inhibited for the 60/40 blend. Since the PBAT did not show biodegradation, biodegradation was more inhibited for the 60/40 blend. Since the PBAT did not show biodegradation, the blends was was highly highly inhibited inhibited at at the the PBAT PBAT layer. the biodegradation biodegradation of of PHBHHx/PBAT PHBHHx/PBAT blends layer. Overall, Overall, these these morphological morphological effects effects cause cause the the decrease decrease of of biodegradability biodegradability in in PHBHHx/PBAT PHBHHx/PBAT sheet. sheet.
(a)
(b)
Figure 4. Typical scanning electron microscope (SEM) micrographs of the blend of PHBHHx/PBAT = Figure 4. Typical scanning electron microscope (SEM) micrographs of the blend of PHBHHx/PBAT = 80/20 (a) before biodegradation. scale bar length is is1 1μm. 80/20 (a) and and 60/40 60/40(b) (b)sheet sheetsurface surface(×5000) (×5000) before biodegradation.The The scale bar length µm.
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(a)
(b)
Figure 5. Typical transmission electron microscope (TEM) micrographs of the cross section of a sheet Figure 5. Typical transmission electron microscope (TEM) micrographs of the cross section of a sheet of (b)(b) before biodegradation. The scale bar length is is 5 μm. of PHBHHx/PBAT PHBHHx/PBAT==80/20 80/20(a) (a)and and60/40 60/40 before biodegradation. The scale bar length 5 µm. Table 5. The effect of morphology of PHBHHx/PBAT blends on the biodegradation in seawater after 28 days. Table 5. The effect of morphology of PHBHHx/PBAT blends on the biodegradation in seawater after 28 days.
Entry Entry 1 12 2 3 3 44 55 66
Mixture Mixture PHBHHx/PBAT PHBHHx/PBAT
(a)
Ratio Ratio 80/20 80/20 80/20 80/20 60/40 60/40 60/40 60/40 40/60 40/60 40/60 40/60
(a)
Form (a) Biodegradation (%) Effect of Morphology Biodegradation (%) Effect of Morphology Form (a) Powder 19 Powder 19 Sheet 10 ○ (b) Sheet 10 # (b) Powder 10 Powder 10 ○ # SheetSheet 2 2 Powder 3 Powder 3 1 degradable SheetSheet 1 NonNon degradable (b)
thickness each sheetsisis20 20 μm; µm; (b) #, waswas observed. The The thickness of of each sheets ○, effect effectofofmorphology morphology observed.
2.5. Effect Effect of the Morphology of PHBHHx/PLA Compound on the Biodegradation Table 6 shows the effect effect of of the the form form of of the the PHBHHx/PLA PHBHHx/PLA blends on their biodegradation in seawater after after2828 days. In these the sheet forms4 and (entries and 6)less also showed less days. In these cases, cases, the sheet forms (entries 6) also4showed biodegradability biodegradability in those comparison with thoseIninthe powder forms. Inthe thePLA/PHA previous paper, the PLA/PHA in comparison with in powder forms. previous paper, biodegradable blends biodegradable blends for pneumothermic fablication of nonwoven was reported [18]. However, there for pneumothermic fablication of nonwoven was reported [18]. However, there is no information on is informationofonPHA the morphology of PHA and PLA the reason for this, scanning thenomorphology and PLA blends. To clarify theblends. reason To forclarify this, scanning electron microscope electron microscope analysis were performed. (SEM) analysis were (SEM) performed. Table 6. 6. The effect of morphology of of PHBHHx/PLA PHBHHx/PLA blends blends on on the the biodegradation biodegradation in seawater after 28 days. Entry 1 12 2 3 3 44 55 66
Entry
Mixture Mixture PHBHHx/PLA
PHBHHx/PLA
(a)
Ratio Ratio 80/20 80/20 80/20 80/20 60/40 60/40 60/40 60/40 40/60 40/60 40/60 40/60
Form (a) (a)Biodegradation (%) Effect of Morphology Biodegradation (%) Effect of Morphology Form Powder 33 Powder 33 Sheet 42 None Sheet 42 None Powder 32 Powder 32 SheetSheet 20 20 None None Powder 26 26 Powder SheetSheet 8 ○ (b)# (b) 8
(b) ○, effect of morphology was observed. (a) The The thickness of of each sheets thickness each sheetsisis20 20 μm; µm; (b) #, effect of morphology was observed.
Figure 6 shows the typical SEM photograph of the sheet surface of the PHBHHx/PLA 80/20 blend Figure 6 shows the typical SEM photograph of the sheet surface of the PHBHHx/PLA 80/20 blend before and after biodegradation in seawater. A lot of hollows were observed on the surface of this before and after biodegradation in seawater. A lot of hollows were observed on the surface of this highly biodegradable sheet after biodegradation (Figure 6b). By contrast, the less biodegradable sheet highly biodegradable sheet after biodegradation (Figure 6b). By contrast, the less biodegradable sheet (60/40) had a relatively flat structure after seawater exposure (Figure 6d). This suggests that the (60/40) had a relatively flat structure after seawater exposure (Figure 6d). This suggests that the surface surface of the less degradable sheet (60/40) was more covered with the low-degradable PLA of the less degradable sheet (60/40) was more covered with the low-degradable PLA component than component than PHBHHx component. Therefore, the masking effect of these blend sheets could be PHBHHx component. Therefore, the masking effect of these blend sheets could be the reason for less the reason for less biodegradability. In conclusion, the surface morphology of the melt blend sheets was an important factor for controlling the biodegradation in seawater.
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biodegradability. In conclusion, the surface morphology of the melt blend sheets was an important factor for controlling the biodegradation in seawater. Mar. Drugs 2018, 16, 34 7 of 11
(a)
(b)
(c)
(d)
Figure 6. SEM photograph (×5000) of the blend sheets of (a) PHBHHx/PLA = 80/20 before, Figure 6. SEM photograph (×5000) of the blend sheets of (a) PHBHHx/PLA = 80/20 before, (b) PHBHHx/PLA PHBHHx/PLA ==80/20 = 60/40 before, (d) (d) PHBHHx/PLA = 60/40 after (b) 80/20after, after,(c)(c)PHBHHx/PLA PHBHHx/PLA = 60/40 before, PHBHHx/PLA = 60/40 biodegradation in seawater. The scale bar length is 1 μm. after biodegradation in seawater. The scale bar length is 1 µm.
Figure 7 shows the surface the fourier-transform infrared spectroscopy (FT-IR) by attenuated Figure 7 shows the surface the fourier-transform infrared spectroscopy (FT-IR) by attenuated total total reflectance (ATR) spectroscopy of sheets of PHBHHx, PLA, and their blends before and after 28 reflectance (ATR) spectroscopy of sheets of PHBHHx, PLA, and their blends before and after 28 days of days of biodegradation in seawater. The peak areas of the C=O stretching bands at 1756 cm−1 biodegradation in seawater. The peak areas of the C=O stretching−1bands at 1756 cm−1 corresponding corresponding to the PLA component and those at 1721 cm corresponding to the PHBHHx to the PLA component and those at 1721 cm−1 corresponding to the PHBHHx component were component were compared before and after biodegradation. As shown in Figure 7, the peak area compared before and after biodegradation. As shown in Figure 7, the peak area corresponding to corresponding to the PHBHHx decreased and the PLA area increased after biodegradation in the PHBHHx decreased and the PLA area increased after biodegradation in seawater. In the case seawater. In the case of PHBHHx/PLA = 60/40 and 40/60, especially, a significant change was of PHBHHx/PLA = 60/40 and 40/60, especially, a significant change was observed in Figure 7c–f. observed in Figure 7c–f. This suggests that the ratio of PLA components increased at the surface of This suggests that the ratio of PLA components increased at the surface of the blend sheets after the blend sheets after biodegradation, indicating that the PHBHHx components degrade biodegradation, indicating that the PHBHHx components degrade preferentially on the surface of preferentially on the surface of PHBHHx/PLA blend sheets. As shown in Table 1, PLA shows higher PHBHHx/PLA blend sheets. As shown in Table 1, PLA shows higher bending elastic modulus than bending elastic modulus than that of PHBHHx. Therefore, the reason of high biodegradation the that of PHBHHx. Therefore, the reason of high biodegradation the PHBHHx/PLA blends powders PHBHHx/PLA blends powders would be caused by the increment of surface of PHBHHx, which is would be caused by the increment of surface of PHBHHx, which is formed some clacking at the freeze formed some clacking at the freeze pulverization process. These increment of the surface area of pulverization process. These increment of the surface area of PHBHHx would be supported by the PHBHHx would be supported by the clear decrease of surface PHBHHx component by FT-IR clear decrease of surface PHBHHx component by FT-IR spectroscopic analyses. spectroscopic analyses.
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Figure 7.7.Fourier-transform Fourier-transform infrared spectroscopy (FT-IR) by attenuated total reflectance (ATR) Figure infrared spectroscopy (FT-IR) by attenuated total reflectance (ATR) spectra spectra of PHBHHx, PLA, theirbefore blendsand before after biodegradation in seawater. of sheetsofofsheets PHBHHx, PLA, and theirand blends afterand biodegradation in seawater.
3. Materials Materials and and Methods Methods 3. 3.1. 3.1. Materials Materials A A commercial commercial grade grade of of PHBHHx PHBHHx (3-HHx (3-HHx unit unit content content == 11 11 mol%, mol%, X151A, X151A, ©KANEKA ©KANEKA Biodegradablepolymer™ Biodegradablepolymer™ PHBH, Kaneka Co., Osaka, Japan) was used in this study. study. PBAT PBAT (Ecoflex (Ecoflex C-1200, (Ingeo 10361D, Nature Works, Minnetonka, MN, USA), PBSand (Bionolle 1020MD, C-1200,BASF), BASF),PLA PLA (Ingeo 10361D, Nature Works, Minnetonka, MN, and USA), PBS (Bionolle Showa Denko, Tokyo, Japan) were used as were obtained. blend (PHBHHx/PBAT, PHBHHx/PLA, 1020MD, Showa Denko, Tokyo, Japan) usedEach as obtained. Each blend (PHBHHx/PBAT, PHBHHx/PBS) prepared bywas a melt extrusion TEM26SS (Toshiba Machine, Numazu, PHBHHx/PLA,was PHBHHx/PBS) prepared byprocess a melt using extrusion process using TEM26SS (Toshiba ◦ Japan) at 140 C with 80 rpm min to obtain Each blended blended pellets. pellets were Machine, Numazu, Japan) at for 1402°C with 80 rpmeach for blended 2 min topellets. obtain each Each pulverized by freeze using a SPEX SamplePre 6750aFreezer/Mill (SPEX) to obtain blend blended pellets werepulverization pulverized by freeze pulverization using SPEX SamplePre 6750 Freezer/Mill powders. average sizeThe of each blend powder was a Microtruc MT3300EXII (SPEX) toThe obtain blendparticle powders. average particle size ofmeasured each blendusing powder was measured using (Nikkiso, Tokyo, Japan). Sheets of theTokyo, blendsJapan). at 20 µm of thickness were also prepared by a T-diewere cast a Microtruc MT3300EXII (Nikkiso, Sheets of the blends at 20 μm of thickness ◦ Ccast extrusion process for 8extrusion min usingprocess a Labo-Plastmill 30C150 (Toyoseiki, Nagano, Japan). also prepared byata 140 T-die at 140 °CModel for 8 min using a Labo-Plastmill Model The data(Toyoseiki, of each bending elastic modulus from the previous [10]. was cited from the 30C150 Nagano, Japan). The was datacited of each bending elasticstudy modulus previous study [10]. 3.2. Biodegradation of Polyesters
3.2. Biodegradation of Polyesters Biodegradation of polyesters with seawater was evaluated using a BOD tester 200 F (TAITEC Co., Tokyo, Japan) according to the with previous reportwas [19].evaluated Seawaterusing was collected at the200 Osaka-Nanko Biodegradation of polyesters seawater a BOD tester F (TAITEC bay area. The typical procedure is as follows: Seawater (200 mL), and sample (30 mg) were placed Co., Tokyo, Japan) according to the previous report [19]. Seawater was collected at the Osaka-Nanko ◦ in a fermenter at 27 procedure C. Ca(OH)is (1.0Seawater g) was used a trap produced CO At the 2 powder 2 gas. bay area. The typical as follows: (200 as mL), and for sample (30 mg) were placed in prescribed time, the amount of consumed O gas was measured. The fermenter, which did not include 2 a fermenter at 27 °C. Ca(OH)2 powder (1.0 g) was used as a trap for produced CO2 gas. At the the sample,time, was the used as a control. The net consumed O2fermenter, gas (mL) which was evaluated as the prescribed amount of consumed O2amount gas wasof measured. The did not include
the sample, was used as a control. The net amount of consumed O2 gas (mL) was evaluated as the difference of the sample and control. The theoretical amount of consumed O2 (mL) means the total
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difference of the sample and control. The theoretical amount of consumed O2 (mL) means the total O2 that should be consumed on complete degradation of sample. Biodegradation (%) was calculated as follows: In the case of PHBHHx (11 mol% of 3-HHx content), the consumption of O2 is the following Equation (1), and the biodegradation is estimated by the Equation (2): C4.22 H6.44 O2 + 4.83 O2 → 4.22 CO2 + 3.22 H2 O,
(1)
Biodegradation (%) = [experimentally consumed O2 (mL)/theoretical O2 (mL)] × 100.
(2)
The consumption of O2 (mL) without samples was deleted from the experimentally consumed O2 (mL). The biodegradation value (%) was average value of duplicate data (N = 2). All biodegradation data using a BOD tester occurred by the microbial metabolism. Therefore, deionized water without microorganisms does not occur with the O2 consumption. 3.3. TEM and SEM Analysis Transmission electron microscope (TEM) observation of each samples were carried out after treatment with RuO4 using a Hitachi H-7650 transmission electron microscope (Hitachi High-Technologies, Tokyo, Japan). Scanning electron microscope (SEM) observation of each samples were also carried out after treatment with RuO4 using Zeiss ULTRAplus scanning electron microscope (Zeiss, Oberkochen, Germany). 3.4. FT-IR Analysis FT-IR spectroscopy of the sample surface by attenuated total reflectance (ATR) spectra was measured using a Perkin Elmer Co., Ltd. (Waltham, MA, USA) model Frontier FT-IR spectrometer. 3.5. 1 H-NMR Analysis 1 H-NMR
analysis of each blends before and after biodegradation was measured using Brucker 400 MHx NMR spectrometer (Bruker Biospin Co., Yokohama, Japan) in CDCl3 as solvent. The content of PHBHHx, PBAT, and PHB was estimated from the following peak areas; PHBHHx = 5.27 ppm (CH), PLA = 5.17 ppm (CH), PBAT = 8.12 ppm (C6 H4 of aromatic protons), PBS = 2.64 ppm (CH2 of succinate). 3.6. Molecular Weight Analysis The molecular weight (Mn, Mw) of PHBHHx, PBAT, PLA, and PBS was determined by GPC (apparatus, Shimadzu LC-10A, Shimadzu Co., Kyoto, Japan, column, Shodex GPCK-806M, Showa Denko, Tokyo, Japan, eluent, chloroform, injection, 10 µL, column temperature, 40 ◦ C) using polystyrene as standard at the concentration of 1.5 mg/mL. 4. Conclusions The PHBHHx sheets and powders showed excellent biodegradability in seawater. The biodegradation of the blends of PHBHHx/PBAT, PHBHHx/PLA, and PHBHHx/PBS was significantly influenced by the weight ratio of their melt blends and decreased in accordance with the decrement of PHBHHx ratio from the BOD tester. The surface morphology of the PHBHHx/PBAT and PHBHHx/PLA blends sheet was an important factor for controlling the biodegradation in seawater based on the SEM and TEM analysis. The PHBHHx component at the surface decreased on degradation in seawater based on the FT-IR (ATR) analysis. From these results, it is clear that the PHBHHx component degraded across a range of blend compositions in seawater. In this study, PHBHHx was shown to have excellent biodegradability in seawater, but the biodegradation rate of other biodegradable polymers was shown to be much lower than that of
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the PHBHHx. From the results of this study, PHBHHx-containing blends degrade much faster than conventional plastics like PBAT, PBS, and PLA and so on, indicating that PHA blends including PHBHHx are useful materials for the marine environment. Supplementary Materials: The following are available online at www.mdpi.com/1660-3397/16/1/34/s1, Figure S1: 1 H-NMR spectra of PHBHHx/PBAT = 80/20, Figure S2-1: 1 H-NMR spectra of PHBHHx/PLA = 80/20, Figure S2-2: Enlarged view of the 1 H-NMR spectra of PHBHHx/PLA = 80/20, Figure S3: 1 H-NMR spectra of PHBHHx/PBS = 80/20, Figure S4: The biodegradation of PHBHHx and PHBHHx/PBS blends (powder shape) in seawater, Figure S5: Particle size distribution of PHBHHx/PLA blend powders. Author Contributions: Hitoshi Sashiwa: Contribution of whole experimental data and whole manuscript preparation, Ryuji Fukuda: Contribution of experimental idea mainly TEM, SEM, and FT-IR analysis and checking manuscript, Tetsuo Okura: Contribution of experimental planning mainly PHBHHx and PBAT, PLA, PBS blends, Shunsuke Sato: Contribution of introduction section mainly biological aspects, Atsuyoshi Nakayama: Experimental contribution of biodegradation using BOD tester. Conflicts of Interest: The authors declare no conflict of interest.
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