Coastal and deep-sea biodegradation of polyhydroxyalkanoate microbeads.

Microbeads find widespread usage in personal care items and cosmetics, serving as exfoliants or scrubbing agents. Their micro-scale size poses challenges in effective drainage capture and given their origin from non-biodegradable oil-based plastics, this contributes substantially to marine pollution. In this study, microbeads were prepared by a simple yet scalable melt homogenization method using four types of polyhydroxyalkanoates (PHA), namely poly[(R)-3-hydroxybutyrate] (P(3HB)), poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyvalerate] (P(3HB-co-3HV)), poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] (P(3HB-co-3HHx)) and poly[(R)-3-hydroxybutyrate-co-(R)-4-hydroxyvalerate] (P(3HB-co-4HB)). Microbeads with different surface smoothness, compressive strength (6.2-13.3 MPa) and diameter (from 1 ~ 150 µm) could be produced. The microbeads were subjected to a comprehensive degradation analysis using three techniques: enzymatic, Biochemical Oxygen Demand (BOD) evaluations, and in situ degradation tests in the deep-sea off Misaki Port in the northern Pacific Ocean (depth of 757 m). Qualitatively, results from enzymatic and in situ degradation demonstrated significant degradation within one week and five months, respectively. Quantitatively, BOD findings indicated that all PHA microbeads degraded similarly to cellulose (~ 85% biodegradability in 25 days). In conclusion, PHA microbeads from this study exhibit promising potential as alternatives to conventional non-biodegradable microbeads.

Microbial decomposition of biodegradable plastics on the deep-sea floor.

Microbes can decompose biodegradable plastics on land, rivers and seashore. However, it is unclear whether deep-sea microbes can degrade biodegradable plastics in the extreme environmental conditions of the seafloor. Here, we report microbial decomposition of representative biodegradable plastics (polyhydroxyalkanoates, biodegradable polyesters, and polysaccharide esters) at diverse deep-sea floor locations ranging in depth from 757 to 5552 m. The degradation of samples was evaluated in terms of weight loss, reduction in material thickness, and surface morphological changes. Poly(L-lactic acid) did not degrade at either shore or deep-sea sites, while other biodegradable polyesters, polyhydroxyalkanoates, and polysaccharide esters were degraded. The rate of degradation slowed with water depth. We analysed the plastic-associated microbial communities by 16S rRNA gene amplicon sequencing and metagenomics. Several dominant microorganisms carried genes potentially encoding plastic-degrading enzymes such as polyhydroxyalkanoate depolymerases and cutinases/polyesterases. Analysis of available metagenomic datasets indicated that these microorganisms are present in other deep-sea locations. Our results confirm that biodegradable plastics can be degraded by the action of microorganisms on the deep-sea floor, although with much less efficiency than in coastal settings.

Thermal Embedding of Humicola insolens Cutinase: A Strategy for Improving Polyester Biodegradation in Seawater.

By thermal embedding of the commercially available enzyme Humicola insolens cutinase (HiC), this study successfully enhanced the biodegradability of various polyesters (PBS, PBSA, PCL, PBAT) in seawater, which otherwise show limited environmental degradability. Melt extrusion above the melting temperature was used for embedding HiC in the polyesters. The overall physical properties of the HiC-embedded films remained almost unchanged compared to those of the neat films. In the buffer, embedding HiC allowed rapid polymer degradation into water-soluble hydrolysis products. Biochemical oxygen demand tests showed that the HiC-embedded polyester films exhibited similar or much higher biodegradability than the biodegradable cellulose standard in natural seawater. Thermal embedding of HiC aims to accelerate the biodegradation of plastics that are already biodegradable but have limited environmental biodegradability, potentially reducing their contribution to environmental problems such as marine microplastics.

In Situ Raman Analysis of Biofilm Exopolysaccharides Formed in Streptococcus mutans and Streptococcus sanguinis Commensal Cultures.

This study probed in vitro the mechanisms of competition/coexistence between Streptococcus sanguinis (known for being correlated with health in the oral cavity) and Streptococcus mutans (responsible for aciduric oral environment and formation of caries) by means of quantitative Raman spectroscopy and imaging. In situ Raman assessments of live bacterial culture/coculture focusing on biofilm exopolysaccharides supported the hypothesis that both species engaged in antagonistic interactions. Experiments of simultaneous colonization always resulted in coexistence, but they also revealed fundamental alterations of the biofilm with respect to their water-insoluble glucan structure. Raman spectra (collected at fixed time but different bacterial ratios) showed clear changes in chemical bonds in glucans, which pointed to an action by Streptococcus sanguinis to discontinue the impermeability of the biofilm constructed by Streptococcus mutans. The concurrent effects of glycosidic bond cleavage in water-insoluble α - 1,3-glucan and oxidation at various sites in glucans' molecular chains supported the hypothesis that secretion of oxygen radicals was the main "chemical weapon" used by Streptococcus sanguinis in coculture.

Marine biodegradation of poly[(R)-3-hydroxybutyrate-co-4-hydroxybutyrate] elastic fibers in seawater: dependence of decomposition rate on highly ordered structure.

Here, we report the marine degradability of polymers with highly ordered structures in natural environmental water using microbial degradation and biochemical oxygen demand (BOD) tests. Three types of elastic fibers (non-porous as-spun, non-porous drawn, and porous drawn) with different highly ordered structures were prepared using poly[(R)-3-hydroxybutyrate-co-16 mol%-4-hydroxybutyrate] [P(3HB-co-16 mol%-4HB)], a well-known polyhydroxyalkanoate. Scanning electron microscopy (SEM) images indicated that microorganisms attached to the fiber surface within several days of testing and degraded the fiber without causing physical disintegration. The results of BOD tests revealed that more than 80% of P(3HB-co-16 mol%-4HB) was degraded by microorganisms in the ocean. The plastisphere was composed of a wide variety of microorganisms, and the microorganisms accumulated on the fiber surfaces differed from those in the biofilms. The microbial degradation rate increased as the degree of molecular orientation and porosity of the fiber increased: as-spun fiber < non-porous drawn fiber < porous drawn fiber. The drawing process induced significant changes in the highly ordered structure of the fiber, such as molecular orientation and porosity, without affecting the crystallinity. The results of SEM observations and X-ray measurements indicated that drawing the fibers oriented the amorphous chains, which promoted enzymatic degradation by microorganisms.

Cleavage of α-1,4-glycosidic linkages by the glycosylphosphatidylinositol-anchored α-amylase AgtA decreases the molecular weight of cell wall α-1,3-glucan in Aspergillus oryzae.

Aspergillus fungi contain α-1,3-glucan with a low proportion of α-1,4-glucan as a major cell wall polysaccharide. Glycosylphosphatidylinositol (GPI)-anchored α-amylases are conserved in Aspergillus fungi. The GPI-anchored α-amylase AmyD in Aspergillus nidulans has been reported to directly suppress the biosynthesis of cell wall α-1,3-glucan but not to degrade it in vivo. However, the detailed mechanism of cell wall α-1,3-glucan biosynthesis regulation by AmyD remains unclear. Here we focused on AoAgtA, which is encoded by the Aspergillus oryzae agtA gene, an ortholog of the A. nidulans amyD gene. Similar to findings in A. nidulans, agtA overexpression in A. oryzae grown in submerged culture decreased the amount of cell wall α-1,3-glucan and led to the formation of smaller hyphal pellets in comparison with the wild-type strain. We analyzed the enzymatic properties of recombinant (r)AoAgtA produced in Pichia pastoris and found that it degraded soluble starch, but not linear bacterial α-1,3-glucan. Furthermore, rAoAgtA cleaved 3-α-maltotetraosylglucose with a structure similar to the predicted boundary structure between the α-1,3-glucan main chain and a short spacer composed of α-1,4-linked glucose residues in cell wall α-1,3-glucan. Interestingly, rAoAgtA randomly cleaved only the α-1,4-glycosidic bonds of 3-α-maltotetraosylglucose, indicating that AoAgtA may cleave the spacer in cell wall α-1,3-glucan. Consistent with this hypothesis, heterologous overexpression of agtA in A. nidulans decreased the molecular weight of cell wall α-1,3-glucan. These in vitro and in vivo properties of AoAgtA suggest that GPI-anchored α-amylases can degrade the spacer α-1,4-glycosidic linkages in cell wall α-1,3-glucan before its insolubilization, and this spacer cleavage decreases the molecular weight of cell wall α-1,3-glucan in vivo.

In Vitro Enzymatic Polymerization of α-1,6-Graft-α-1,3-glucan and Structural Analysis of Gel Formation.

α-1,6-Graft-α-1,3-glucan comprises a main-chain of α-1,6-glucan and side-chains of α-1,3-glucan. It was synthesized by a one-pot in vitro enzymatic polymerization of sucrose and dextran (α-1,6-glucan) of different molecular weights. In the presence of the high-molecular-weightdextran (Mw ≥ 650 000), the graft glucan formed a self-standing hydrogel without any cross-linker. It was possible to control the number of α-1,3-glucan side-chains by controlling the molecular weight and concentration of the dextran. Consequently, it was possible to control the compression strength of the obtained gels. Hydrogels of the graft glucan were formed by physically cross-linking the α-1,3-glucan side-chains. These physical gels are potentially useful biomaterials with high biocompatible, because the graft glucan is composed of glucose alone.

Enzymatic control and evaluation of degrees of polymerization of ß-(1→2)-glucans.

ß-(1 â†’ 2)-Glucans can be synthesized by 1,2-ß-oligoglucan phosphorylase using ß-(1 â†’ 2)-glucooligosaccharides as acceptors and α-d-glucose 1-phosphate as a donor. Using phosphorolysis of sucrose as a source of α-d-glucose 1-phosphate, we generated ß-(1 â†’ 2)-glucans with degrees of polymerization (DPs) up to approximately 280. Average DPs up to approximately 1000 were obtained using ß-(1 â†’ 2)-glucan with average DP of 160 as an acceptor and pure α-d-glucose 1-phosphate as a donor. A colorimetric assay of the ß-glucosidase activity against the ß-(1 â†’ 2)-glucan products was used to determine their DPs.

High Tensile Strength Regenerated α-1,3-Glucan Fiber and Crystal Transition.

α-1,3-Glucan is a linear and crystalline polysaccharide which is synthesized by in vitro enzymatic polymerization from sucrose. A previous study reported that regenerated fibers of α-1,3-glucan were prepared using a wet-spinning method. However, the mechanical properties were poorer than cellulose regenerated fibers. Then, in this study, the mechanical properties of the regenerated α-1,3-glucan fiber were improved by the transformation of the crystal structure and stretching. The regenerated fiber stretched in water and dehydrated by heating showed high tensile strength (18 cN/tex) that is comparable with that of viscose rayon. Moreover, the crystal structures of the regenerated fibers were investigated using wide-angle X-ray diffraction (WAXD). To date, four crystal polymorphs of α-1,3-glucan from polymorph I to IV have been reported. This study revealed that the regenerated α-1,3-glucan fibers had two different polymorphs, polymorph II (hydrated form) and polymorph III (anhydrous form), depending on post-treatment methods of stretching and annealing procedures. Furthermore, the obtained distinctive 2D-WAXD patterns suggested that polymorph III is identical to polymorph IV.

Highly stretchable curdlan hydrogels and mechanically strong stretched-dried-gel-films obtained by strain-induced crystallization.

Highly-stretchable self-standing curdlan (1,3-ß-d-glucan) hydrogels were prepared via chemical cross-linking using various cross-linkers, including ethylene glycol diglycidyl ether, 1,4-butandiol diglycidyl ether, and 1,6-hexanediol diglycidyl ether. Tensile testing of the curdlan hydrogels revealed that the hydrogels had good elongation properties with 600%-900% elongation strain from their original length regardless of the cross-linker length. Stretched-dried-gel films were prepared by stretching of the hydrogels and subsequent drying. The tensile strength and Young's modulus of the stretched-dried-gel-films were 117-148 MPa and 1.6 GPa, respectively, and these values were markedly improved compared with the non-stretched films. X-ray measurements revealed that the stretched dried-gel films had oriented crystalline domains with an 80% of degree of orientation. These results indicate that the curdlan molecular chains were oriented and crystallized during the process of stretching and drying of the hydrogels. As a result, the stretched-dried-films showed a high tensile strength owing to strain-induced crystallization.

Mechanism of Elastic Properties of Biodegradable Poly[(R)-3-Hydroxybutyrate-co-4-hydroxybutyrate] Films Revealed by Synchrotron Radiation.

Reversible elastic films of biobased and biodegradable poly[(R)-3-hydroxybutyrate-co-4-hydroxybutyrate] [P(3HB-co-4HB)] were prepared by uniaxial drawing procedures. Mechanical properties and highly ordered film structures were investigated by tensile testing and both static-state and in situ wide-angle X-ray diffraction and small-angle X-ray scattering with synchrotron radiation during stretching and relaxing. Despite the crystalline nature of the polymers, the elongation at break of these films was greater than 1500%. Reversible elasticity was achieved after the first 10 times of uniaxial stretching. X-ray measurement results indicated that on stretching, ß-form molecular chains with a planar zigzag conformation were introduced from molecular chains with random coils in the amorphous regions between α-form lamellar crystals. Notably, the orientation of the α-form lamellar crystals increased after relaxation of the molecular chains with a planar zigzag conformation (ß-form) between the lamellar crystals (α-form). Reversible elastic properties were regenerated by a planar zigzag conformation between the lamellar crystals, the extension of molecular chains in lamellar crystals by the rotation of molecular conformation, and changes in the degree of orientation of the lamellar crystals.

Highly deformable and recoverable cross-linked hydrogels of 1,3-α-d and 1,3-ß-d-glucans.

We prepared self-standing chemically cross-linked hydrogels from 1,3-α-d-glucan (Mw = 2.0 × 105) and 1,3-ß-d-glucans (low-molecular-weight (LMW): Mw = 2.0 × 105, high-molecular-weight (HMW): Mw = 1.0 × 106), using ethylene glycol diglycidyl ether (EGDGE) as a cross-linker. Uniaxial compressive tests using cylindrical hydrogels of the cross-linked glucans were conducted. Both the 1,3-α-d-glucan and LMW-1,3-ß-d-glucan hydrogels were highly deformable and shape-deformable; they could be compressed without breaking to 60% and 80% strain, respectively, and recovered 80% of their original height. The Young's moduli of the 1,3-α-d-glucan and LMW-1,3-ß-d-glucan hydrogels indicated that the 1,3-α-d-glucan hydrogels were harder than the 1,3-ß-d-glucan hydrogels. The HMW-1,3-ß-d-glucan hydrogels were more deformable and had better shape recovery than the LMW-1,3-ß-d-glucans; they could be compressed by up to 90% maximum strain, and recovered almost 100% of their original height from 80% strain. Cyclic compression tests were performed to study their network structure.

Comparative Studies on Regioselectivity of α- and ß-Linked Glucan Tosylation.

Alpha- and beta-linked 1,3-glucans have been subjected to conversion with p-toluenesulfonic acid (tosyl) chloride and triethylamine under homogeneous reaction conditions in N,N-dimethyl acetamide/LiCl. Samples with a degree of substitution of tosyl groups (DSTs) of up to 1.91 were prepared by applying 5 mol reagent per mole repeating unit. Hence, the reactivity of α-1,3-glucan is comparable with cellulose and starch, while the ß-1,3-linked glucan curdlan is less reactive. The samples dissolve in aprotic dipolar media independent of the DSTs and possess a solubility in less polar solvents that depends on the DSTs. NMR studies on the tosyl glucans and of the peracylated derivatives showed a preferred tosylation of position 2 of the repeating unit. However, the selectivity is less pronounced compared with starch. It could be concluded that the α-configurated glycosidic bond directs tosyl groups towards position 2.

Primary structure of gum arabic and its dynamics at oil/water interface.

Gum arabic (GA), an arabinogalactan-based gum, is a well-known powerful emulsifier. However, the poor stability of emulsion has often been pointed out. In order to clarify the origin, the structure-property relationship of GA, especially the interfacial property at oil/water interface, needs to be investigated. Here, we tried to correlate the primary structure with interfacial property at oil/water interface. A series of structural analyses by SEC-MALLS, SAXS, etc. showed that the primary structure of GA was a disk-like star shaped nanoparticle. The dynamic interfacial tension measurement showed that GA molecules adsorb onto oil surface in 2 steps: Firstly, the micron-aggregates of GA approach onto the oil surface, and then the aggregates are dissociated into nano-particles so that they cover the oil surface. Therefore, the emulsification and emulsion stability are controlled not by the property of the primary structure of GA but by the higher-order molecular network structure made of GA molecules.

Enzymatic Self-Biodegradation of Poly(l-lactic acid) Films by Embedded Heat-Treated and Immobilized Proteinase K.

Non-biodegradable microplastics have become a global problem. We propose a new enzyme-embedded biodegradable plastic that can be self-biodegraded anytime and anywhere. Proteinase K from Tritirachium album was embedded in poly(l-lactic acid) (PLLA). The PLLA solution-cast film with embedded proteinase K showed weight loss of 78% after 96 h incubation. In addition, PLLA extruded films embedding immobilized proteinase K encapsulated in polyacrylamide were produced at 200 °C and embedded-enzyme degradation was monitored. Immobilized proteinase K embedded in the extruded film maintained its degradation activity and degraded the PLLA film from inside to make small holes and cavities, suggesting that immobilization is a powerful technique to prepare thermoforms with embedded enzymes. The rate of embedded-enzyme degradation was accelerated by dividing the film into smaller pieces, which can be regarded as a model experiment for biodegradation of microplastics. Various biodegradable plastics with specific embedded enzymes will contribute to solve global environmental problems.

Large-scale preparation of ß-1,2-glucan using quite a small amount of sophorose.

A large amount of ß-1,2-glucan was produced enzymatically from quite a small amount of sophorose as an acceptor material through three synthesis steps using a sucrose phosphorylase and a 1,2-ß-oligoglucan phosphorylase. The first synthesis step was performed in a 200 µL of a reaction solution containing 5 mM sophorose and 1.0 M sucrose. ß-1,2-Glucan in a part of the resultant solution was hydrolyzed to ß-1,2-glucooligosaccharides by a ß-1,2-glucanase. The second synthesis was performed in 25 times the volume for the first synthesis. The hydrolysate solution (1% volume of the reaction solution) was used as an acceptor. After treatment with the ß-1,2-glucanase again, the third synthesis was performed 200 times the volume for the second synthesis (1 L). The reaction yield of ß-1,2-glucan at each synthesis was 93%, 76% and 91%. Finally, more than 140 g of ß-1,2-glucan was synthesized using approximately 20 µg of sophorose as the starting acceptor material. Abbreviations: DPs: degrees of polymerization; SOGP: 1,2-ß-oligoglucan phosphorylase; Sopns: ß-1,2-glucooligosaccharides with DP of n; Glc1P: α-glucose 1-phosphate; SucP: sucrose phosphorylase from Bifidobacterium longum subsp. longum; SGL: ß-1,2-glucanase; CaSGL: Chy400_4174 protein; TLC: thin layer chromatography; GOPOD: glucose oxidase/peroxidase; PGM: phosphoglucomutase; G6PDH: glucose 6-phosphate dehydrogenase.

Recyclable Fully Biobased Chitosan Adsorbents Spray-Dried in One Pot to Microscopic Size and Enhanced Adsorption Capacity.

A facile one-pot spray-drying process was developed for fabrication and in situ crosslinking of chitosan microspheres to improve the adsorption capacity by microscopic design. A fully biobased nature was achieved by utilizing genipin (GP) as a crosslinking agent and chitosan-derived nanographene oxide (nGO) as a property tuner. The produced chitosan microspheres were further proven as powerful adsorbents for common wastewater contaminants such as anionic dyes and pharmaceutical contaminants, here modeled by methyl orange (MO) and diclofenac sodium (DCF). By regulating the amount of GP and nGO, as well as by controlling the process parameters including the spray-drying inlet temperature and postheat treatment, the surface morphology, size, zeta potential, and adsorption efficiency of the microspheres could be tuned accordingly. The adsorption efficiency for MO and DCF reached 98.9 and 100%, respectively. The microspheres retained high DCF adsorption efficiency after six adsorption and desorption cycles, and the recyclability was improved by the incorporated nGO. The fabricated microspheres, thus, have great potential as reusable and eco-friendly adsorbents.

Synthesis and Characterization of Biobased Polyesters Containing Anthraquinones Derived from Gallic Acid.

To study the effects of the main and side chains on the physical properties of polyesters containing anthraquinone substituents, two types of 1,5-dihydroxy-2,3,6,7-tetraalkoxyanthraquinones (DHTAAQs) were prepared from gallic acid, a major component of hydrolyzable tannins, and polymerized with five dicarboxylic dichlorides by interfacial polymerization. The solubility of the fabricated polyesters was strongly affected by the alkoxy side chains of the DHTAAQ units. Conversely, their thermal decomposition behavior depended on the structure of the dicarboxylate units. The thermal properties of the anthraquinone-based polyesters were influenced by both the dicarboxylate units and alkoxy groups, and their glass transition temperature could be controlled over a wide range (81-308 °C). To design DHTAAQ-based polyesters with adequate solubility and high heat resistance, it is preferable that first suitable alkoxy groups are selected to provide the necessary solubility, and then appropriate aromatic dicarboxylate units are chosen to obtain the required thermal properties.

Lipase-Catalyzed Regioselective Synthesis of Dextrin Esters.

Four lipase enzymes were investigated as catalysts in the synthesis of regioselectively monosubstituted dextrin esters from dextrin and vinyl acetate. An immobilized lipase enzyme (Lipozyme TL IM) exhibited the highest activity. This enzyme showed regioselective substitution of the dextrin at the primary hydroxyl group (C6 position) under optimal conditions (60 °C for 24 h, using a 1:3 molar ratio of glucose unit/vinyl acetate and 2.5 U/mL enzyme dosage in an organic solvent). To compare the reactivity of other vinyl esters, monosubstituted dextrin esters (degrees of substitution [DS] ≈ 1) with varying side-chain lengths (C2-C12) were synthesized. With increasing side-chain length, the initial catalytic activity of the lipase enzyme decreased, resulting in lower DS values. However, the final DS values of the monosubstituted dextrin esters with longer side chains were higher than those of the shorter-chain analogues, because of an increase in affinity between the substrate and acyl donor.

Molecular Mass and Localization of α-1,3-Glucan in Cell Wall Control the Degree of Hyphal Aggregation in Liquid Culture of Aspergillus nidulans.

α-1,3-Glucan is one of the main polysaccharides in the cell wall of filamentous fungi. Aspergillus nidulans has two α-1,3-glucan synthase genes, agsA and agsB. We previously revealed that AgsB is a major α-1,3-glucan synthase in vegetative hyphae, but the function of AgsA remained unknown because of its low expression level and lack of phenotypic alteration upon gene disruption. To clarify the role of α-1,3-glucan in hyphal aggregation, we constructed strains overexpressing agsA (agsAOE ) or agsB (agsBOE ), in which the other α-1,3-glucan synthase gene was disrupted. In liquid culture, the wild-type and agsBOE strains formed tightly aggregated hyphal pellets, whereas agsAOE hyphae aggregated weakly. We analyzed the chemical properties of cell wall α-1,3-glucan from the agsAOE and agsBOE strains. The peak molecular mass of α-1,3-glucan from the agsAOE strain (1,480 ± 80 kDa) was much larger than that from the wild type (147 ± 52 kDa) and agsBOE (372 ± 47 kDa); however, the peak molecular mass of repeating subunits in α-1,3-glucan was almost the same (after Smith degradation: agsAOE , 41.6 ± 5.8 kDa; agsBOE , 38.3 ± 3.0 kDa). We also analyzed localization of α-1,3-glucan in the cell wall of the two strains by fluorescent labeling with α-1,3-glucan-binding domain-fused GFP (AGBD-GFP). α-1,3-Glucan of the agsBOE cells was clearly located in the outermost layer, whereas weak labeling was detected in the agsAOE cells. However, the agsAOE cells treated with ß-1,3-glucanase were clearly labeled with AGBD-GFP. These observations suggest that ß-1,3-glucan covered most of α-1,3-glucan synthesized by AgsA, although a small amount of α-1,3-glucan was still present in the outer layer. We also constructed a strain with disruption of the amyG gene, which encodes an intracellular α-amylase that synthesizes α-1,4-glucooligosaccharide as a primer for α-1,3-glucan biosynthesis. In this strain, the hyphal pellets and peak molecular mass of α-1,3-glucan (94.5 ± 1.4 kDa) were smaller than in the wild-type strain, and α-1,3-glucan was still labeled with AGBD-GFP in the outermost layer. Overall, these results suggest that hyphal pellet formation depends on the molecular mass and spatial localization of α-1,3-glucan as well as the amount of α-1,3-glucan in the cell wall of A. nidulans.