Influence of mold temperature on the properties of wastewater-grown microalgae-based plastics processed by injection molding

1 Influence of mold temperature on the properties of wastewatergrown microalgae-based plastics processed by injection molding R. M. González-Balderasab, M. Felixb, C. Bengoecheab, A. Guerrerob, M.T. Orta Ledesmaa*. aInstituto de Ingeniería, Coordinación de Ingeniería Ambiental, Universidad Nacional Autónoma de México UNAM, Circuito Escolar s/n, Ciudad Universitaria, Delegación Coyoacán, México, CDMX., C.P. 04510. bDepartamento de Ingeniería Química, Universidad de Sevilla, Escuela Politécnica Superior, 41011, Sevilla, Spain. *Corresponding author at: Instituto de Ingeniería, Coordinación de Ingeniería Ambiental, Universidad Nacional Autónoma de México UNAM, Circuito Escolar s/n, Ciudad Universitaria, Delegación Coyoacán, México, CDMX., C.P. 04510, Emails: [email protected]. 2 Keywords Bioplastics; Desmodesmus sp.; Microalgal proteins; Tetradesmus obliquus; Wastewater Abbreviations ANOVA____ Analysis of variance DMTA____Dynamic mechanical thermal analysis DSC____Differential scanning calorimetry GL____glycerol LSM____loss of soluble material LVR____Linear viscoelastic region PHAs____Polyhydroxyalkanoates PLA____Polylactic acid SEM____Scanning electron microscopy WUA____Water uptake capacity G'____Elastic moduli G''____Viscous moduli tan δ____Loss tangent E____Young's module εmax____Strain at break 3 σmax____Maximum stress 4 Abstract Nowadays there is a need for sustainably produced degradable bio-based plastics to lessen the high dependence on non-renewable petrochemical resources. Therefore, this study evaluates the development of bio-based plastics from Desmodesmus sp. and Tetradesmus obliquus biomass cultivated in wastewater. Also, it evaluates the effects of mold temperature over the mechanical and viscoelastic properties of the bioplastics by dynamical mechanical analysis (DMA), tensile tests, and water uptake capacity (WUC). Results prove that an increase in injection molding temperature resulted in an increase in the viscoelastic properties (elastic moduli, G', from oscillatory torsion tests increased from 3.8 · 107 ± 0.2 Pa to 6.0 ± 0.1 Pa and from 2.2 · 107 ± 0.2 Pa to 3.3 ± 0.1 Pa for Desmodesmus sp. and T. obliquus, respectively) and a decrease in WUC (from 865.9 ± 44.6 % to 447.5 ± 28.7 %, and from 393.6 ± 18.6 % to 219.2 ± 5.3 % for Desmodesmus sp. and T. obliquus, respectively), suggesting a direct relationship between protein cross-linking. Desmodesmus sp. based bioplastics showed higher maximum stress and WUC than T. obliquus based bioplastics, while T. obliquus based plastics showed higher strain at break. These differences could be related to the high cell wall rigidity of Desmodesmus sp. which might induce the formation of lower toughness probes with higher swelling capacity. Moreover, its higher protein content (31%) may lead to stronger molecular interactions which explained a higher σmax (28.8 MPa). On the other hand, T. obliquus induced compact, rigid, and tough plastics that kept their structure after water immersion. Additionally, a certain cell wall disruption may take place when processing T. obliquus based plastic, which may favor cross-linking to a larger extent than in Desmodesmus sp. system. The bio-based plastic materials developed may provide high-value and sustainable solutions for the by-products generated in wastewater treatment. 5 1. Introduction Plastics are produced within a profitable and growing industry whose global production was 359 Mt in 2018, being mostly used in the packaging sector (39.9 %) [1]. However, plastics represent a serious environmental burden because they are low recyclable and recoverable (around 20%). Also, microorganisms present in nature have not evolved to efficiently degrade petroleum-derived polymers, resulting in a negligible biodegradability [2]. Therefore, the development of sustainably produced degradable bio-based plastics is being promoted, which additionally would lessen the high dependence on non-renewable petrochemical resources. Most bio-based plastics, such as polylactic acid (PLA) or polyhydroxyalkanoates (PHAs), are nowadays produced from carbohydrates and plant materials. However, there is a concern that, as bio-based plastics are increasingly demanded, natural carbon cycles could be interfered and alter soil fertility [3]. The so-called next generation microalgae systems could help to alleviate those issues, as they tune into a preferred circular bioeconomy. Moreover, they are non-food competitive, locatable on non-arable land, and they can use wastewater for recycling of nutrients such as nitrogen and phosphorous. This use can result in the reduction of eutrophication and dependence on chemical fertilizers [3]. Desmodesmus and Tetradesmus species are indigenous microalgae a primarily components in plankton [4]. Both species of microalgae possess a high protein content, even comparable to commercial cyanobacteria largely found in the food market [5]. Also, Desmodesmus and Tetradesmus are commonly applied to wastewater treatment through nutrient removal and simultaneously valorization of microalgae biomass for biofuel production [6, 7]. The cultivation of microalgae in wastewater treatment plants affects their morphology and biochemical composition, as it depends on the medium in which the cells are growing. However, from an economic and environmental perspective, microalgae-based wastewater treatment is 6 considered a promising alternative to bacterial-based processes [6, 8]. Moreover, the resulting microalgae biomass could be revalorized through their use in the manufacture of bio-based plastics. Due it has already been proven that microalgae-based plastics may be processed by traditional thermoplastic polymer processing techniques such as injection molding [9]. The use of injection molding is of great importance due to its easily scalable character, which would enable larger productions in a more environmentally friendly way [10]. Microalgae biomass is an attractive raw material to produce bioplastics since they are protein-rich materials (ranging from 20 to 71 wt% on a dry basis). Also, the high the protein percentage made them with a great similarity to petrochemical plastics [9]. The use of microalgae biomass for bioplastics ranges from its direct use as bioplastic to its blending with petrochemical and other biobased plastics [11-12]. In this sense, Zeller et al. [13] studied the obtention of bioplastics based on commercial Spirulina platensis and Chlorella vulgaris plasticized with glycerol in different ratios (0-30 wt%). They determined that a microalgae:glycerol ratio of 80:20 produced the best material properties. All bioplastic samples were processed by thermomechanical molding for 20 min at 150 °C. In a more recent study, López Rocha et al. [12] obtained bioplastics from a microalgae consortia plasticized with glycerol. All bioplastics obtained displayed a glass transition temperature around 60 °C. The bioplastics showed a low ability to absorb water when immersed, due to the low deformability displayed in the tensile tests. Due to the need for sustainable biodegradable bioplastic materials, the present study aims the development and characterization of plastics based on Desmodesmus sp. and Tetradesmus obliquus biomass cultivated in wastewater. Additionally, it evaluates the effects of mold temperature over the mechanical and viscoelastic properties of the bioplastics measured by dynamical mechanical analysis (DMA) and tensile tests, as well as on the water uptake capacity. 7 2. Material and methods 2.1. Microalgae biomass and materials The microalgae Desmodesmus sp. used in this study were native microalgae isolated from the artificial lake ‘‘Lago Nabor Carrillo’’, located in Texcoco, Mexico, to which an effluent from facultative lagoon wastewater treatment plant was poured. The morphology of Desmodesmus sp. was characterized via light microscopy observations with the aid of identification manuals [14]. The species identified were Desmodesmus intermedius (Chodat) Hegewald, Desmodesmus magnus (Meyen) Tsarenko, Desmodesmus communis (Hegewald) Hegewald and Desmodesmus opoliensis (Richter) Hegewald. The microalgae, Tetradesmus obliquus (Turpin) Kützing, used in this study was provided by Instituto Politecnico Nacional, Mexico City, Mexico. T. obliquus was previously adapted to municipal wastewater. Both Desmodesmus sp. and T. obliquus biomass were grown in 10 L reactors at room temperature (20 ± 3 °C). Microalgae species were inoculated with an inoculum size of 300 ± 14 mg/L. To ensure consistency on the microalgae protein content, cultures were fed every 7 days during 27 days using municipal wastewater (85 ± 16 mg/L of NH4+- N, 544 ± 5.5 mg/L of COD, 46 ± 4.5 mg/L of ortho-PO4-3, and 47 ± 1.5 mg/L of total phosphorus). Culture pH was maintained between 7 and 8 to control bacterial growth. The light was provided by LED lamps (1620 lumen), under light/dark periods of 12:12 h. The biomass was harvested by sedimentation; the unwashed biomass was oven dried at 40 ℃ for 12 h and stored at 4 ℃ until used, two separate samples were obtained. The approximate composition of each biomass sample was determined by cell counts using a Neubauer counting chamber and microscope (AX10 Lab A1, Zeizz, Jena, Deutschland). The Desmodesmus sp. and T. obliquus microalgae cultures were respectively composed of 90% Desmodesmus sp., 5% Cyanobacteria sp., and 5% Mychonastes sp., and 90% Tetradesmus obliquus, 5% Cyanobacteria sp., and 5% Mychonastes sp. To ensure 8 consistency all experiments were done using the same batch of each species of microalgae biomass. Glycerol (GL) was purchased from Panreac Química (Spain) and used as plasticizer. 2.2. Microalgae protein content Protein content was determined as % N×6.25 using a LECO CHNS-932 nitrogen micro analyzer (Leco Corporation, St. Joseph, MI, USA). Measurements were performed at least in triplicate. 2.3. Bioplastics preparation Bioplastic materials were obtained following a two-stage method. Firstly, microalgae biomass (Desmodesmus sp. and T. obliquus) and plasticizer (GL) were blended into a mixer in an appropriate protein/plasticizer ratio (60/40) [9]. The mixer used was a two-blade counter-rotating rheometer (Haake Polylab QC, ThermoHaake), which permits the control of both temperature and torque values. Dough-like blends were obtained after 5 min mixing at 25 °C and 50 rpm. Polymer/plasticizer blends were then submitted to injection molding processing to obtain the bioplastics probes. A lab scale injection molding machine MiniJet II (ThermoHaake, Germany) was used. Rectangular specimens (60×10×1mm) were obtained using an injection pressure of 500 bar for 20 s, and a holding pressure of 200 bar for 200 s. The temperature of the injection chamber was 60 °C in all cases, whereas three different mold temperatures were tested (80, 100, and 120 °C). At least ten probes for each mold temperature were obtained. 2.4. Differential scanning calorimetry (DSC) DSC measurements were performed on biomass using a Mettler Toledo DSC822, provided with a robotic-arm and with an electric intercooler as a refrigeration unit (Mettler Toledo S.A.E., 9 Barcelona, Spain). Samples (4.0 ± 0.1 mg) were hermetically encapsulated in aluminum pans to prevent mass losing during heating from -50 to 250 °C at a rate of 10 °C/min under inert atmosphere conditions (10 mL/min N2). 2.5. Rheological characterization of blends Dough-like blends were characterized by small amplitude oscillatory shear (SAOS) measurements, using a controlled-strain rheometer (AR-2000, TA Instruments, USA). The rheometer was equipped with a plate–plate geometry (dia.: 40 mm) with a rough surface, selecting a gap between plates of 2 mm. Elastic moduli (G'), viscous moduli (G''), and loss tangent (tan δ) were obtained through small amplitude shear oscillatory (SAOS) tests. In every case, strain sweep tests were first carried out to determine the linear viscoelastic region (LVR). Subsequently, viscoelastic properties were measured through dynamic assays at 1 Hz at different temperatures. All these measurements were carried out at constant strain amplitude (0.2%) within the LVR. Measurements were performed at least in triplicate. 2.6. Characterization of bioplastics 2.6.1. Dynamic mechanical thermal analysis (DMTA) Rheological characterization of the final probes was carried out by means of DMTA tests, which were performed using a Discovery DHR3 rheometer (TA Instruments, USA). Torsion tests were performed to rectangular geometry probes. Frequency sweep tests were carried out from 0.02 to 20 Hz at constant temperature (30 °C). Moreover, temperature ramp tests from -30 to 140 °C at 3 °C/min and at 1 Hz were performed. Both tests were carried out within the linear viscoelastic region. At least triplicate measurements were performed. 2.6.2. Tensile tests 16 Table 1 Viscoelastic response at 1 Hz (G'1 and G''1) as a function of frequency obtained for the Desmodesmus sp. and Tetradesmus obliquus/glycerol mixtures at four different mold temperatures: 25, 80, 100 and 120 °C. System Temperature G'1 (Pa) G''1 (Pa) tan δ1 Desmodesmus sp. 25 °C 1.8 · 105 ± 3.5 · 104 4.1 · 104 ± 3.5 · 104 0.29 ± 1.5 · 10-2 80 °C 1.5 · 106 ± 9.0 · 104 4.3 · 105 ± 4.4 · 101 0.33 ± 2.2 · 10-2 100 °C 1.8 · 106 ± 8.4 · 104 5.4 · 105 ± 9.0 · 104 0.30 ± 1.8 · 10-2 120 °C 1.5 · 106 ± 1.5 · 105 3.5 · 105 ± 2.6 · 104 0.26 ± 2.7 · 10-2 Tetradesmus obliquus 25 °C 3.5 · 105 ± 2.2 · 104 1.1 · 105 ± 4.4 · 104 0.29 ± 5 · 10-3 80 °C 1.1 · 105 ± 4.6 · 104 3.5 · 104 ± 1.9 · 103 0.32 ± 1.3 · 10-2 100 °C 9.5 · 105 ± 6.4 · 104 2.3 · 105 ± 1.4 · 104 0.24 ± 8.7 · 10-3 120 °C 9.0 · 105 ± 1.7 · 104 2.2 · 105 ± 5.2 · 104 0.24 ± 7 · 10-3 Data given as average values with standard error of the mean (n=3). None of the factors (microalgae species and mold temperature) have a statically significant effect on the viscoelastic response of the biomass/glycerol mixtures at the 95% level. 17 3.3 Characterization of bioplastics 3.3.2. Thermal-mechanical-dynamic analysis (DMTA) Fig. 3 shows viscoelastic moduli (G' and G'') and loss tangent (tan δ) as a function of temperature, for Desmodesmus sp. (Fig. 3a) and T. obliquus (Fig. 3b) based plastics. This figure shows a similar behavior for both systems, being the elastic component (G') always above the viscous (G''). Moreover, the thermal profile of G' passes through two inflection points as it decreases continuously as temperature increases for all the systems obtained, which would permit the detection of two glass transition temperatures, Tg1 and Tg2 [18]. The value for those glass transition temperatures may be defined through the maximum found in the tan δ profile [22], although it is not clear for all samples, as sometimes those peaks seem to overlap. Tg1 and Tg2 are located around 10 and 90 ºC, respectively, for Desmodesmus sp., and around 5 and 60 ºC for T. obliquus. Previously, other authors have reported glass transition temperatures around 70 ºC when studying Spirulina and Chlorella plastics, or 60 ºC for a Scenedesmus sp. and Desmodesmus sp. consortia based plastic [9, 13]. The fact that no strengthening is observed at higher temperatures for any sample indicates that neither of them displays any thermosetting potential at the selected experimental conditions. Thus, neither higher temperatures nor longer residence times are required during the injection molding processing to reinforce the samples [23]. Table 2 shows the elastic moduli (G'1), the viscous moduli (G''1), and the loss tangent (tan δ1) at 1 Hz for all the bioplastics studied. In Desmodesmus sp. based plastics, the highest G'1 values are obtained for the sample molded at 120 °C, which would indicate that a higher mold temperature reinforced the system, suggesting that hydrophobic and disulfide interactions are formed in a greater extent then and that mold temperature is always lower than the 18 degradation temperature of the protein system [18, 19, 24]. Therefore, Desmodesmus sp. based bioplastics show greater viscoelastic properties than T. obliquus based bioplastics, which agrees with previous results obtained for the corresponding blends. Fig. 3. Viscoelastic response (G', G'', and loss tangent) as a function of temperature for a) Desmodesmus sp. and b) Tetradesmus obliquus based bioplastics at three different mold temperatures: 80, 100 and 120 °C. -50 050 100 150 10 6 10 7 10 8 10 9 G', G''/(Pa) Temperature (°C) G' G'' tan δ Tmold 80 °C 100 °C 120 °C -50 050 100 150 Temperature (°C) G' G'' tan δTmold 80 °C 100 °C 120 °C 0.0 0.1 0.2 0.3 tan δ 19 Table 2 Viscoelastic response (G'1, G''1, and tan δ1) at 1 Hz as a function of frequency obtained for Desmodesmus sp. and Tetradesmus obliquus based bioplastics at three different mold temperatures: 80, 100 and 120 °C. System Tmold (°C) G'1 (Pa) G''1 (Pa)a tan δ1 Desmodesmus sp. 80 °C 3.8 · 107 ± 2.3 · 106 9.3 · 106 ± 5.4 · 10 0.24 ± 5.1 · 10-4 100 °C 4.9 · 107 ± 2.0 · 106 1.2 · 107 ± 3.3 · 106 0.23 ± 2.5 · 10-3 120 °C 6.0 · 107 ± 6.2 · 105 1.4 · 107 ± 1.5 · 105 0.23 ± 4.8 · 10-3 Tetradesmus obliquus 80 °C 2.2 · 107 ± 2.3 · 106 5.4 · 106 ± 6.7 · 105 0.24 ± 4.8 · 10-3 100 °C 3.6 · 107 ± 2.5 · 106 8.3 · 106 ± 9 · 105 0.23 ± 9.2 · 10-3 120 °C 3.3 · 107 ± 1.2 · 106 7.9 · 106 ± 2.8 · 105 0.24 ± 2.9 · 10-5 Data given as average values with standard error of the mean (n=3). aMicroalga species have a statically significant effect on G'' response for the bioplastics at the 95% level. aP-value 0.049. 20 3.3.3. Tensile measurements Fig. 4 shows the stress-strain curves obtained from uniaxial strength measurements performed to Desmodesmus sp. and T. obliquus based bioplastics. The profiles obtained follow the typical pattern of other polymeric and protein-based bioplastic materials [18]. All curves have an initial linear region, with a constant tension slope -deformation (corresponding to Young's Modulus, E). Then, there is a long plastic region, with a continuous decrease in the stress-strain slope towards a constant value; after the elastic limit, the maximum stress (σmax) and strain at break (εmax) are reached, which eventually leads to the rupture of the probe [23]. Stress-strain curves for all bioplastics indicated that the increase in mold temperature induces an increase in σmax and εmax. Table 3 summarizes the main mechanical parameters (E, σmax, and εmax) obtained from the stressstrain curves of the uniaxial tensile strength tests. Generally, samples become more resistant and deformable (higher σmax and εmax) as mold temperature increases. Moreover, greater rigidity (E) and resistance (σmax) are displayed by the Desmodesmus sp. based bioplastics, which possesses a higher protein content. Thus, the Desmodesmus sp. based plastics show σmax values two orders of magnitude greater than those presented by the T. obliquus based plastics. On the other hand, the T. obliquus based bioplastics are more deformable, showing εmax values one order of magnitude greater than the corresponding Desmodesmus sp. based bioplastics. The greater rigidity presented by Desmodesmus sp. samples could also be attributed to the different rigidity of cell walls for both types of microalgae. Desmodesmus sp. cells have four sporopollenin wall layers with submicroscopic ornaments on the outer layer, whereas sporopollenin has an aliphatic polymer that forms crosslinks between straight-chain aliphatic compounds, phenolic and aromatic groups. Additionally, most Desmodesmus species have one or more spines or "teeth" in each cell, while these structures are absent in Tetradesmus. On the other hand, T. obliquus cells exhibit a 21 characteristic trilaminar structure [25] that contains cellulose in the inner wall layers and algaenans in the trilaminar layer [26]. Proteins, especially highly glycosylated forms, are common and important constituents of microalgae cell walls. Hydroxyproline-rich glycoproteins, also called extensins, have been the protein most frequently found in the microalgae cell wall. Extensins selfassemble to form scaffolds on the cell wall, help maintain wall structure, improve tensile strength, and contribute to plaque formation and in defense responses [27]. Also, extensins are known to be among the key components responsible for cell wall rigidification as a result of cross-linking with each other and with other wall components including wall polysaccharides, signaling molecules, and possibly receptors on plasma membranes [28]. Therefore, it is likely that reactions between cell wall proteins induce strong interactions in spite of exhibits low toughness. The influence of the microalga species on the mechanical properties of the bioplastics is confirmed by the Multifactor ANOVA analysis. Since the results showed that the microalga species has a statically significant effect on E and σmax. In the Desmodesmus sp. based bioplastics, σmax and εmax increase as mold temperature increases. This information suggests that temperature-induced protein cross-linking promotes the formation of more fragile probes in comparison to those based on T. obliquus [29]. In this respect, Lagrain et al. [30], stated that increasing the elongation at the break of glassy, amorphous polymers typically goes at the expense of the elastic modulus. 22 Fig. 4. Stress-strain curves for a) Desmodesmus sp. and b) Tetradesmus obliquus based bioplastics at three different mold temperature (80, 100 and 120 °C). 0.00 0.01 0.02 0.03 0.04 0.0 0.2 0.4 5 10 15 20 25 30 σ (MPa) ε (mm/mm) 80 °C 100 °C 120 °C Desmodesmus sp. Tetradesmus obliquus T mold 23 Table 3 Tensile parameters, Young’s modulus (E), maximum stress (σmax) and strain at break (εmax), obtained for Desmodesmus sp. and Tetradesmus obliquus based bioplastics as a function of mold temperature (80, 100 and 120 °C). Data given as average values with standard error of the mean (n=3). a,bMicroalga species have a statically significant effect on E and σmax obtained for the bioplastics at the 95% level. subP-value: a0.018 and b0.035. System T mold E (MPa)a σ max (MPa)b εmax (%) Desmodesmus sp. 80 °C 198.3 ± 6.3 9.5 ± 1.0 0.33 ± 0.02 100 °C 112.8 ± 3.5 15.8 ± 1.6 0.28 ± 0.01 120 °C 100.7 ± 10.6 28.7 ± 2.3 0.56 ± 0.07 Tetradesmus obliquus 80 °C 11.6 ± 0.6 0.14 ± 0.01 0.33 ± 0.02 100 °C 24.2 ± 2.5 0.33 ± 0.03 3.2 ± 0.34 120 °C 21.2 ± 2.0 0.35 ± 0.09 4.8 ± 0.21 24 3.3.4. Water uptake capacity (WUC) Fig. 5 shows values for WUC and LSM for Desmodesmus sp. (Fig. 5a) and T. obliquus (Fig. 5b) based bioplastics after 24 hours of water immersion of bioplastic samples. The results show that WUC decreases with increasing mold temperature for all protein systems studied, which can be explained by the increase in cross-linking between protein molecules as the mold temperature increases since it prevents the specimens from swelling [31, 32]. Additionally, it is important to highlight that the WUC depends on the microalgae system (Desmodesmus sp. or T. obliquus), where the Desmodesmus sp. based bioplastics show 2-fold WUC than the T. obliquus based bioplastics. On the other hand, although the LSM appears to be around 50% by weight in all the bioplastic materials obtained, the increase in mold temperature leads to a slight decrease in this value. The increase in protein cross-linking as mold temperature increases has been reported to lead to a further decrease in the loss of soluble material [31, 32]. However, as the glycerol content was always kept at 40 %, the results obtained would imply that apart from the hydrophilic plasticizer, the biomass sample is partially loss (~10 %). 80 100 120 0 200 400 600 800 1000 Water uptake capacity (%) Tmold (°C) Water uptake capacity (WUC) Loss of soluble matter (LSM) 80 100 120 T mold (°C) Water uptake capacity (WUC) 0 20 40 60 Loss of soluble matter (LSM) Loss of soluble matter (%) 25 Fig. 5. Water uptake capacity (WUC, %) after 24 h water immersion and loss soluble matter (LSM, %) for a) Desmodesmus sp. and b) Tetradesmus obliquus based bioplastics as a function of mold temperature (80, 100 and 120 °C). 3.3.5. Scanning electron microscopy (SEM) Fig. 6 shows SEM images obtained for bioplastic materials after 24 h water immersion. SEM images show that the probes keep a more compact and seemingly rigid structure as mold temperature increased. Thus, at 80 ºC, matrices resulting from freeze drying the swelled plastics display a more porous microstructure when compared to those obtained from the samples molded at 120 ºC. These results agree with previous results, which indicated that the increase in mold temperature induced an increase in protein crosslinking and consequently a decrease in water uptake. Moreover, Desmodesmus sp. matrices display pores of greater size than the corresponding T. obliquus matrices which could also be attributed to the different rigidity of cell walls for both types of microalgae. As was previously indicated Desmodesmus sp. cells have a cell wall with four sporopollenin wall layers whilst T. obliquus cells display a cell wall with trilaminar structure [25] that only contains cellulose and algaenans [26]. This fact is important because sporopollenin is an aliphatic polymer that forms crosslinks between straight-chain aliphatic compounds which increase the rigidity of microalgae cell walls. 32 B. 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