Biodegradation of chemically modified lignocellulosic sisal fibers: study of the mechanism for enzymatic degradation of cellulose

2.1 Soil burial test

A significantly modified method was adopted over an existing soil burial test (SBT) to evaluate the long-term biodegradation resistance of untreated and treated fibers. The compost burial experiment was prepared by mixing black organic garden soil, sand, and cow dung in a 2:1:1 ratio (by weight) according to the method reported by the Bureau of Indian Standards (BIS) (6). The weight of the fibers was taken to be about 1% of the total weight of the compost. The fibers were buried in the compost in a glass enclosure following the method reported elsewhere (14). All the glass enclosures were stored in an incubator at 30±2°C and 85±5% relative humidity. The biodegradation test was performed for 120 days, and the compost was replaced with a fresh formulation once every week to simulate an open biodegradation environment that is likely to be observed outdoors. The present test protocol differs from existing biodegradation test method that recommends soil burial test to continue only for 21 days, and it involves no periodic replacement of compost during the test. A similar procedure was also described in the ASTM G160 standard (5) using horse manure in place of cow dung; however, the standard does not suggest any replenishment of soil/compost mixture either. The laboratory-based soil/compost mixture used in this test provides more aggressive degradation environments compared to outdoor field exposure (15). About six representative tests in six different glass enclosures were conducted for each batch of fiber sample. Soil pH was maintained in between 6 and 7 throughout the test duration by adding a dilute solution of hydrochloric acid or sodium hydroxide periodically. The moisture content of the soil was controlled around 65±5% over the entire test process.

2.2 Enzymatic activities

The sisal fibers periodically recovered during SBT were washed with sterilized distilled water in a rotating mechanical shaker at 180 rpm for 1 h. The washed liquor was then centrifuged at 6000 rpm for 10 min at 4°C. The final supernatant solution was used for assessing cellulase, xylanase, and lipase activities using the protocols presented in the next few subsections.

2.3 Microbial population count

The microbial population within the test compost before and during the biodegradation exposure was regularly counted after every 30 days using the plate dilution technique. Eight-fold serial dilutions of each compost sample were prepared in sterilized distilled water, and about 0.1 ml of the diluted sample isolated from the compost solution was spread on the surface of a nutrient agar medium consisting of 0.3% beef extract, 0.5% peptone, 0.5% NaCl, and 1.7% agar with pH of ∼7.0. The agar medium was kept for 24-h incubation at 35°C. The colony on the plate surface was counted, and microbial population count (MPC) was reported as the number of colony forming units per gram of compost.

2.4 Chemical composition of fibers

Gravimetric analysis was performed to estimate the percentage of chemical constituents of the untreated and oil-resin-treated sisal fibers. In order to execute this experiment, about 1 g of fiber sample was refluxed with about 10 ml of neutral detergent solution (NDS) in a round-bottom flask with successive addition of 2 ml of decahydronaphthalene and 0.5 g of sodium sulfate for 1 h at 70–80°C. The residue was collected upon filtering with a grade 2 sintered glass crucible. Washing of the residue was performed sequentially with hot water and acetone followed by drying at 100°C for 8 h to remove the water-soluble constituents of the fiber sample, such as pectin. Similarly, 1 g of fiber was boiled with 100 ml of acid detergent solution for 5–10 min and refluxed for 1 h at 70–80°C to remove the water-soluble fiber constituents such as hemicelluloses. Again, the second type of residue collected by filtering through the G2 crucible was washed first with hot water and then with acetone before drying at 100°C for 8 h. The amount of hemicellulose was estimated by obtaining the weight difference of the two residues (19). The residue obtained from the acid detergent treatment was stirred with 50 ml of 72% sulfuric acid with 1 g of asbestos granules (3 mm long) for 3 h in a beaker to remove lignin. The residue was washed, filtered through Whatman No. 1 filter paper, and dried at 100°C for 2 h. The weight of the residue from this operation represented an estimate of the cellulose content of the fiber sample. Transesterified cellulose chains of the oil-resin-treated fibers were also included in this quantification; however, the loss of weight was considered as an estimate of the lignin content of the fibers.

2.5 Crystallinity

X-ray diffractograms of untreated and oil-resin-treated fiber samples were obtained before and after biodegradation. Powdered samples of the fibers (1±0.5 g) were subjected to X-ray diffraction (XRD) on a RIGAKU, Japan (ULTIMA III) instrument operated at room temperature using Cu-Kα source (λ=1.54 nm) and at a scanning speed of 2°/min. The observed ranges of 2θ were found between the 10° and the 50° regions. From the XRD diffractogram, a plot between apparent intensity and 2θ was evaluated to estimate the crystallinity index (CrI) using the following equation:

where I_22.5 and I_18.5 are the intensities at 2θ values of 22.5° and 18.5°, respectively. Detailed evaluation of the data presented by other researchers (20) indicates that the CrI for each sample can be used as an approximate measure of the content of crystalline cellulose if multiplied by a factor of 0.78 (weight divided by fiber dry weight) for LCFs.

2.6 Infrared spectroscopy

Chemical changes in the fiber samples as a result of biodegradation were assessed by comparing their FTIR spectra taken before and after the degradation. The relative abundance of fiber surface-bound hydroxyl groups that were most susceptible to biodegradation was also assessed from the FTIR spectra. Infrared spectra were obtained for wave numbers ranging between 4000 and 400 cm^-1 using 32 scans for each sample. The amount of each fiber sample was taken to be about 1±0.2 mg, which was thoroughly mixed with 100±10 mg of potassium bromide (KBr) to form a pellet. The infrared spectra were obtained by keeping the pellet samples on a Bruker Tensor 27 (Bruker UK Ltd.) instrument operated at 35±2°C. Peaks representing different functional groups in the FTIR spectrum were evaluated after dividing the intensity of each peak with the intensity at 1060 cm^-1, as proposed by Colom et al. (21).

2.7 Water absorption and tensile strength

Water absorption of the fibers before SBT was estimated by testing in triplicate fiber samples of 10 g (dry mass) following the ASTM D 570-98 standard (22). The mean and 95% confidence intervals of these parameters are reported.

A single fiber strand separated from a fiber bundle under an optical microscope was pasted into a cardboard frame with a rectangular window of 15 mm in width and 25 mm in height using an adhesive, as shown in Figure 1. Before the separation of each fiber strand, the fiber bundles were moisture conditioned at a relative humidity of 65±2% and a temperature of 21±1°C for 24 h according to the ASTM D 1776 standard (23). The frame with a fiber strand was placed between two jaws of a universal testing machine (UTM; Hounsfield H10KS, Tinius Olsen Ltd., UK) with a 100-N load cell attachment. Least count for load range least count of UTM was 5N. Load was applied to the fiber using a crosshead speed of 3 mm/min. For each batch of fiber, the tensile strength of 50 fiber samples was measured according to the ASTM D 3822 standard (24) using the average fiber diameter. The mean of the 50 measurements is reported in this study.

Figure 1:

Cardboard frame for mounting single fibers for measuring the tensile strength.

2.8 Surface morphology

The surface morphology of the fibers after 120 days of biodegradation was evaluated through scanning electron microscopy (SEM). About 1–2 mg of an LCF sample, 1–2 mm in length, was washed with dilute acetone and oven dried at 80±5°C. The samples were exposed to a plasma sputtering apparatus to coat them with a thin gold layer. The coated surfaces were examined with a TESCAN VEGALSV, Czech Republic, scanning electron microscope in high-vacuum mode with a voltage of between 5 and 10 kV.

3.1 Enzymatic degradation profile of LCFs

During the initial 30 days of SBT, cellulase and xylanase activities were found to increase significantly before declining sharply (see Figure 2A and B). This initial rapid increase in enzymatic activities was found to be more prominent and higher for oil-resin-treated fibers than for untreated fibers. The reason might be due to the substantial removal of lignin and waxes from fiber matrices during transesterification reaction in an alkaline environment. A rapid enzymatic hydrolysis took place during this phase owing to the absence of the shielding effect provided by less biodegradable lignin and wax molecules. This phase of biodegradation was dominated by the degradation of mainly amorphous cellulose with virtually no degradation of crystalline cellulose.

Figure 2:

(A) Cellulase, (B) xylanase, and (C) lipase activities during LCF biodegradation. Cellulase, xylanase, and lipase activities were closely related to the degradation of cellulose, hemicelluloses, and aryl/acyl ester of newly formed ester bond in treated fibers. (D) Degradation profile of treated and untreated fiber with biodegradation exposure.

Cellulase activity continued to decline sharply after 30 days for the oil-resin-treated fibers and after 45 days for the untreated fibers. The lack of accessible amorphous cellulose for the microorganisms within the oil-resin-treated fibers possibly induced the faster decline in cellulase activity. However, cellulase activity was found to again increase abruptly owing to the onset of degradation of crystalline cellulose for both the untreated and the oil-resin-treated fibers beyond 60 days of exposure. An increasing trend of cellulase activity continued up to 120 days for the untreated fibers, whereas in the case of oil-resin-based treatment the cellulase activity was found to decline after 90 days. This interesting observation indicates the less accessibility of crystalline cellulose for the treated fibers due to the applied chemical modification for enhanced durability. An increase in cellulase activity after 90 days up to 120 days for the untreated fibers was observed due to the facilitated availability of unmodified cellulose molecules for degradation by microorganisms. A slight decreasing trend of microbial population after 90 days for the untreated fibers was further correlated with increasing cellulase activity. Initial cellulase activity (up to 30 days) for both untreated and oil-resin-treated fibers was found to be almost comparable. The reason might be due to the rapid initial degradation of accessible cellulose of some unmodified cellulose part in the oil-resin-treated fibers. However, with degradation exposure time the cellulase activity for the untreated fibers was found to be more significant than that for the oil-resin-treated fibers. With a degradation time beyond 30 days, it was evident that the increase in cellulase activity for the untreated fibers was found to be almost 1.5 times than that for the oil-resin-treated fibers.

During the last 75 days (for oil-resin-treated sisal fibers; Figure 2B) and 90 days (for the untreated sisal fibers; Figure 2B) of the testing period, xylanase activity exhibited a steady decrease. That indicates a continuous depletion or non-availability of hemicelluloses within fiber matrices after the initial aggressive degradation profile.

For oil-resin-treated fibers, lipase activity registered an intense increase during the initial few days and declined rapidly thereafter in the SBT (Figure 2C). The initial increase in lipase activity supports the onset of a hydrolytic degradation of acyl side chains that were incorporated into the fiber matrix during the transesterification reaction. In contrast, the lipase activity for the untreated fibers declined rapidly starting from an initial value similar to that for the oil-resin-treated fibers (Figure 2C). Thereafter the decline was less rapid. These observations indicate the relative non-availability of lipolytic constituents within the untreated sisal fibers.

The overall observation and degradation profile of each constituent of untreated and oil-resin-treated fibers are presented in Figure 2D.

3.2 Microbial population

The enzymatic activities and MPC estimated in this study (Table 1) were substantially higher than those reported in other studies (25, 26). As a result, the modified soil burial protocol performed in this study provided more severe condition for fiber samples compared to other existing standard protocols. For soil/compost mixtures containing oil-resin-treated sisal fibers, the microbial population was found to be fewer compared to those containing untreated fibers throughout the 120-day soil biodegradation test (Figure 3). Consequently, oil-resin-treated sisal fibers are expected to exhibit lower susceptibility to microbial attack, leading to the occurrence of a smaller microbial population. Some of the explanations for the declining microbial population over the initial 30–45 days of biodegradation are the delayed fiber swelling and lag phase in microbial population growth. The subsequent increase in microbial population continuing over a period of up to 90 days possibly corresponds to the population growth triggered by the use of LCF constituents as nutrients. The microbial population beyond 90 days was found to decrease sharply due to the unavailability of degradable cellulose in treated fiber matrices.

Figure 3:

Changes in microbial population with duration of biodegradation.

In contrast, untreated LCFs exhibited a greater tendency to attract water molecules through H-bond formation with accessible OH groups, causing the fibers to swell, resulting in the subsequent biodegradation in the presence of larger microbial population. Beyond 90 days, microbial population became stabilized for the untreated sisal fibers and started to decline steadily for the oil-resin-treated sisal fibers, possibly because of depletion of nutrients in the form of celluloses or hemicelluloses. Unlike the microbial population of oil-resin-treated fibers, that of untreated fibers remained almost unchanged even after 90 days of exposure. The possible reason might be the availability of amorphous cellulose in the untreated fibers.

3.3 Changes in fiber chemistry

The estimated amount of residual percentage of cellulose (amorphous and crystalline), hemicellulose, and lignin during the biodegradation exposure for both untreated and oil-resin-treated fibers is presented in Figure 4A–D. The crystalline cellulose was measured from the data obtained from XRD analysis, whereas the residual percentage of other constituents was measured by gravimetric chemical analysis as described in the experimental sections. The data presented in Figure 4 for residual fiber components throughout the biodegradation exposure support the proposed biodegradation profile presented earlier in Figure 2D. The initial extent of cellulose, hemicellulose, and lignin before biodegradation was also measured and is presented in Table 2.

Figure 4:

Impact of biodegradation on the chemical composition such as (A) total cellulose, (B) crystalline cellulose, (C) hemicelluloses, and (D) lignin for lignocellulosic fibers.

Figure 5 shows the FTIR intensity at 1734 cm^-1 (region “A”) for the untreated sisal fibers, which represents the C=O stretching vibration of aryl ester of hemicellulose and lignin (27). The intensity of this peak was reduced remarkably following SBT, which, in turn, indicates the substantial hydrolytic removal of hemicellulose and lignin. The relatively insignificant impact of SBT on FTIR spectral response in the same region or in the oil-resin-treated fibers, representing the C=O stretching vibration of acyl ester of the acyl side chains incorporated during transesterification, further indicates the resistance against the enzymatic hydrolysis of newly formed fatty ester bonds of the treated fibers.

Figure 5:

FTIR spectra of untreated and oil-resin-treated fibers throughout the SBT. (“A” and “B” in the Figure indicate two specific regions around 1734 and 1250 cm^-1 respectively)

Similarly, the FTIR spectral response around 1250 cm^-1 indicated by region B for untreated sisal fiber in Figure 5 represents the stretching vibration of C–O–C and C–O of the ester groups of lignin (28) and hemicelluloses (29), prominently weakened throughout the SBT, indicating the continuous enzymatic degradation of the amorphous materials. The corresponding reduction of peak intensity of SBT for the oil-resin-treated fibers in region “B” was somewhat less pronounced, indicating a relative inertness of the partly transesterified ester bonds within the treated fibers. The observation seen in FTIR spectra supports the data presented in Figure 4 on the relative changes in residual content of celluloses (both crystalline and amorphous), hemicelluloses and lignin during SBT.

3.4 Changes in physical properties

Table 2 indicates that the untreated fibers absorbed more water molecules compared to the oil-resin-treated fibers; therefore they are expected to show more intense swelling. Generally, higher surface swelling of untreated sisal fibers is expected to accelerate the enzymatic degradation of hemicelluloses and cellulose macromolecules. Because of the tight packing of crystalline cellulose chains, the hygroscopic hydroxyl groups of crystalline cellulose are not as susceptible to biodegradation as those of hemicelluloses and amorphous cellulose (30). As a result, crystalline cellulose is expected to exhibit lower swelling tendencies and greater resistance against biodegradation during the initial phase of biodegradation.

The relative unavailability of residual lignin and hemicelluloses in the oil-resin-treated fibers and the reduced swelling tendency of partially transesterified amorphous as well as crystalline cellulose molecules resulted in higher resistance to biodegradation for these fibers compared to the untreated ones. Therefore, oil-resin-treated fibers retained a greater proportion of tensile strength after SBT compared to untreated fibers (Figure 6A). The effect of microbial degradation in soil/compost mixture without replacement was also observed for 120 days. It was seen that the severity of the degradation was much more significant in soil/compost mixture replaced periodically. In order to show the effect of acid and alkaline solution used during SBT to maintain the soil pH on the tensile strength of fibers, a control test in dilute HCl (0.5% by volume) and 0.5% NaOH solution (by volume) was also carried out. The results are presented in Figure 6B. It can be seen that the effect of dilute acid and alkali solution on the tensile strength of the fibers was marginal. In order to confirm the better resistance of treated fibers against biodegradation, the surface morphology of both untreated and treated fibers was also observed in photomicrographs obtained from SEM. A drastic erosion of the fiber surface was observed in the case of untreated fibers after SBT (Figure 7). The surfaces of oil-resin-treated fibers were found to be less affected during the 120 days of biodegradation. It can be inferred that the application of chemical treatment on fibers (oil-resin-treated fibers for instance) results in greater resistance to biodegradation and hence less reduction of tensile strength owing to the microbial exposure during SBT.

Figure 6:

(A) Retention of the tensile strength of untreated and oil-resin-treated fibers throughout the SBT with and without changing the soil composite. (B) Retention of the tensile strength of untreated and oil-resin-treated fibers throughout the SBT in HCl and NaOH medium.

Figure 7:

Effect of the SBT on the surface morphology of untreated and oil-resin-treated fibers.