Effects of alkaline or liquid-ammonia treatment on crystalline cellulose: changes in crystalline structure and effects on enzymatic digestibility

Abstract

Background: In converting biomass to bioethanol, pretreatment is a key step intended to render cellulose more amenable and accessible to cellulase enzymes and thus increase glucose yields. In this study, four cellulose samples with different degrees of polymerization and crystallinity indexes were subjected to aqueous sodium hydroxide and anhydrous liquid ammonia treatments. The effects of the treatments on cellulose crystalline structure were studied, in addition to the effects on the digestibility of the celluloses by a cellulase complex.

Results: From X-ray diffractograms and nuclear magnetic resonance spectra, it was revealed that treatment with liquid ammonia produced the cellulose IIII allomorph; however, crystallinity depended on treatment conditions. Treatment at a low temperature (25°C) resulted in a less crystalline product, whereas treatment at elevated temperatures (130°C or 140°C) gave a more crystalline product. Treatment of cellulose I with aqueous sodium hydroxide (16.5 percent by weight) resulted in formation of cellulose II, but also produced a much less crystalline cellulose. The relative digestibilities of the different cellulose allomorphs were tested by exposing the treated and untreated cellulose samples to a commercial enzyme mixture (Genencor-Danisco; GC 220). The digestibility results showed that the starting cellulose I samples were the least digestible (except for corn stover cellulose, which had a high amorphous content). Treatment with sodium hydroxide produced the most digestible cellulose, followed by treatment with liquid ammonia at a low temperature. Factor analysis indicated that initial rates of digestion (up to 24 hours) were most strongly correlated with amorphous content. Correlation of allomorph type with digestibility was weak, but was strongest with cellulose conversion at later times. The cellulose IIII samples produced at higher temperatures had comparable crystallinities to the initial cellulose I samples, but achieved higher levels of cellulose conversion, at longer digestion times.

Conclusions: Earlier studies have focused on determining which cellulose allomorph is the most digestible. In this study we have found that the chemical treatments to produce different allomorphs also changed the crystallinity of the cellulose, and this had a significant effect on the digestibility of the substrate. When determining the relative digestibilities of different cellulose allomorphs it is essential to also consider the relative crystallinities of the celluloses being tested.

Figure 1

X-ray diffractograms showing the effect of temperature and hold time during liquid ammonia treatment on conversion of cellulose I to cellulose III_I. (a) 140°C, (b) 130°C, (c) 105°C, (d) 25°C for 90 min, (e) 25°C for 5 min, and (f) -33°C for 15 min, (g) the Avicel PH 101 control cellulose I, (h) is amorphous cellulose.

Figure 2

X-ray diffractograms. (A) untreated cellulose I, (B) cellulose I treated with NaOH to make cellulose II, (C) cellulose I treated with ammonia at 130°C to make cellulose III_I, from different cellulose samples. (a) cotton linters, (b) Avicel PH 101, (c) α-cellulose, (d) cellulose isolated from corn stover, (e) amorphous cellulose.

Figure 3

¹³C CPMAS solid-state NMR spectra. (A) Original cellulose I and (B) cellulose II for various cellulose samples. (a) Avicel, (b) α-cellulose, (c) cotton linters, and (d) corn stover, (e) amorphous cellulose. CPMAS: cross polarization and magic angle spinning solid-state; NMR: nuclear magnetic resonance.

Figure 4

¹³C CPMAS solid-state NMR spectra of cellulose III_I prepared by treatment with liquid ammonia. (A) At 25°C and (B) 130°C from various cellulose I samples. (a) Avicel, (b) α-cellulose, (c) cotton linters, and (d) corn stover, (e) amorphous cellulose. CPMAS: cross polarization and magic angle spinning solid-state; NMR: nuclear magnetic resonance.

Figure 5

Molecular weight distributions of Avicel PH 101 and treated Avicel samples.

Figure 6

Enzymatic hydrolysis of different cellulose samples. (a) Avicel PH 101, (b) α-cellulose, (c) cotton linters, (d) corn stover cellulose. (The error bars indicate the reproducibility (± 1 standard deviation) of digestions conducted in triplicate on the Avicel cellulose samples).

Figure 7

Comparison of cellulose conversion levels after (a) 16 h, (b) 24 h, (c) 48 h, and (d) 72 h for the starting cellulose I, NaOH treated, NH₃ treated at 25°C, and NH₃ treated at 130°C. (Note: The highly digestible, high amorphous content, corn stover cellulose I sample is connected to the other cellulose I samples by a dashed line).

Figure 8

Factor analysis of cellulose conversion levels at different digestion times (16 h to 72 h) with amorphous content, initial cellulose type, and allomorph type. This chart shows the correlations between variables and factors after Varimax rotation.

Figure 9

Temperature profile for the liquid ammonia treatment conducted at 25°C for 5 min.