Abstract

Determination of tissue proportion and vascular bundle morphology

To soften the samples, the fixed blocks were soaked in a thermostat water bath operated at 60°C for 2 weeks. Transverse-sections with a thickness of 20 μm were cut from the softened sample blocks by using a sliding microtome (Leica RM2010R, Wetzlar, Germany). The intact slices were randomly selected and dehydrated by freeze-drying. To analyze the morphological characteristics of the vascular bundles and the proportion of the cell tissue, the dehydrated transverse-sections were coated with gold and then observed using a field-emission scanning electron microscope (XL30-FEG-SEM, FEI, USA; Fig. 1c). The tissue proportion was calculated using the square grid method (Qi et al. 2013). After the transparent paper with a square grid was pasted onto the computer screen, the fibrous, ground, and conducting tissues were counted using the square grid method to calculate the tissue proportion, and 15 image fields were selected for each sample.

Determination of fiber morphology

The remaining transverse-sections were stained with 0.1 percent safranin within 10 minutes, dehydrated with an alcohol series, and mounted in Canada balsam until permanent slices were finished (Fig. 1d). Subsequently, 30 measurements were determined for the fiber lumen diameter and fiber double-cell-wall thickness. The fixed blocks were rinsed several times with deionized water and artificially chipped into sticks (2 by 2 by 10 mm). The sticks were then macerated in a mixture of 30 percent hydrogen peroxide and glacial acetic acid (1:1 vol/vol) at 60°C under stirring until the separation of the fibers from their matrix (Hisham et al. 2006; Fig. 1e). The separated fibers were immediately transferred onto the glass slides to determine their length and width; 70 fibers from each sample were examined. Fiber lengths were measured in accordance with the standard applied for papermaking (Code of China 2011). The parameters of the fiber were observed using a digital microscope camera (DMLB2, Leica, Germany), and the quantitative data for the outer, middle, and inner parts across the culm wall were recorded separately.

Statistical analysis

The slenderness ratio and Runkel ratio of the fiber were calculated based on Equation 1 and Equation 2, where L is the length of the fiber, D is the width of the fiber, 2w is the thickness of the double cell wall, and d is the lumen width of the fiber:

In addition, the experimental data were statistically analyzed and compared by analysis of variance (ANOVA) using the least significant difference method to determine the level of significance at P ≤ 0.05.

Frequency of vascular bundle

The vascular bundle morphologies of B. pervariabilis culms at different heights are presented in Figure 3. In accordance with the vascular bundle classification by Liese (1998), two vascular bundle types—the broken-waist type and the semiundifferentiated type—were observed from the bottom zone to the top zone of B. pervariabilis culms. The typical vascular bundle morphology is classified as the broken-waist type, consisting of a central vascular strand and a fiber strand. The transverse-section of the bamboo culms consists of numerous vertical oval-shaped vascular bundles that are smaller near the periphery and then become sparser and larger toward the middle of the culm. Meanwhile, the inner layer consists of vascular bundles that are widely arranged and transformed into oval-shaped bundles arranged sideways (Fig. 3; Table 1). No significant differences in the frequency of the vascular bundles were observed in all age groups. However, the vascular bundle of bamboo culms in the Age 2 group was more concentrated than those in the Age 1 and Age 2 groups. Similar trends in Bambusa vulgaris were described by Wahab et al. (2010). The distribution of vascular bundles from the bottom to the top in all age groups exhibited an increasing trend. The vascular bundles with a more concentrated distribution near the top endow the top of the culm with excellent mechanical strength (Grosser and Liese 1971, Liese 1998, Londoño et al. 2002, Wang et al. 2016).

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Table 1 Characteristics of the vascular bundles in B. pervariabilis at different ages and heights.^a

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Size of vascular bundle

No significant differences in the radial and tangential widths of the vascular bundles were found between the samples with different ages (Table 1). Both radial and tangential widths in the Age 2 group were less than those in the Age 1 and Age 3 groups. From the bottom to the top of the culm, the width sharply decreased, with the radial width decreasing significantly more acutely than the tangential width. Similar trends were reported by Hisham et al. (2006), Kelemwork (2009), and Wahab et al. (2010).

Length, width, and slenderness ratio of fibers

Compared with a short fiber, a long fiber provides higher tensile strength and tearing strength (Zhan et al. 2015). Fiber length in the 1,448.91- to 2,410.94-μm range, with an average of 1,953.30 μm, is considered to be that of a long fiber. The morphology of the fiber and statistical results are presented in Figure 4 and Table 2. There was a slight decrease in length with age, and this variation was mainly attributable to the maturation of bamboo in the Age 1 group (Nordahlia et al. 2012). Fiber length also varied significantly with culm height, and the largest mean (1.99 mm) was observed in the middle of the culms (Table 2). This finding is consistent with previous studies (Wang et al. 2011, Zhan et al. 2015) in which fiber length was affected by internode length, and the middle internodes were longer than the bottom and top internodes.

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Table 2 Fiber characteristics (length, width, and slenderness ratio) of B. pervariabilis fibers with at different ages and heights.^a

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Fiber width significantly affected the crossover area of the fiber. Generally, a wider fiber presents a larger crossover area per fiber, which is favorable for the production of paper with improved quality and strength (Anupam et al. 2016). Age did not significantly affect fiber width, and the largest mean was measured in the Age 2 group (Table 2). This observation was consistent with the report by Wang et al. (2011) that fibers complete their width growth in 1 or 2 years. From the bottom to the top of the culm, the mean width of fiber gradually and significantly narrowed, which may be caused by the tapering structure of bamboo culms (Table 2).

The length-to-width ratio of the fiber is also known as the slenderness ratio of the fiber. The pulp obtained from wood with thin-walled fibers provides dense and well-bonded sheets, whereas that from wood with thick-walled fibers produces bulky sheets with high tear resistance (Anupam et al. 2016). In this study, the slenderness ratio of the B. pervariabilis fiber ranged from 104.24:1 to 163.79:1, with an average of 140.01:1. This average is higher than that of wood fibers, indicating that fiber exhibits superior tensile strength and tear resistance. The age of the bamboo culms exerted no significant effect on the slenderness ratio of the fiber. However, there was significant difference between slenderness ratios by height, whereby the minimum mean was observed in the bottom of the culm (Table 2).

Wall thickness, lumen diameter, and Runkel ratio.—The measurements of the double-wall thickness, lumen diameter, and Runkel ratio of the fiber are summarized in Table 3. The wall thickness ranged from 7.04 to 8.75 μm, and the lumen diameter varied from 2.29 to 3.05 μm. Age exerted a significant effect on the wall thickness and lumen diameter of the bamboo fiber. As the age of the bamboo culm increased, the cell wall thickness generally increased, whereas the lumen diameter decreased owing to the formation of a multilayered structure and lignin deposition in the cell wall. The height of the bamboo culm significantly influenced the wall thickness but not the lumen diameter of the fiber, as shown by the sharp decrease in cell wall thickness from the bottom to the top of the bamboo culm, as well as slight narrowing of the lumen diameter of the fiber.

Table 3 Fiber characteristics (double-wall thickness, lumen diameter, and Runkel ratio) of B. pervariabilis fibers with different ages and heights.^a

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The wall-to-lumen ratio, also known as the Runkel ratio, was determined based on the double-wall thickness to lumen diameter, which is one of the important indexes for assessing the softness and interweaving of the fiber (Anupam et al. 2016). Significant differences in Runkel ratio were found between the bamboo samples of different age groups, with the average ranging from 2.56 to 3.02 and abruptly increasing from the Age 1 group to the Age 3 group (Table 3). Moreover, the Runkel ratio was not significantly affected by the height of the bamboo culm; the bottom had the largest ratio. According to previous reports, the fiber Runkel ratio is preferably < 1 to obtain good pulp raw materials (Yang et al. 2008). The mean ratio indicated that the suitability of bamboo for pulp raw materials was inferior to those of hardwood and softwood.