4.4. Low-moisture anhydrous ammonia pretreatment
(LMAA)
It has been known that cellulose chains are incorporated
into a number of distinct crystal phases each differing in
chemical reactivity and material characteristics. The naturally
occurring crystal phases, cellulose I [52], can be transformed
into cellulose IIII by treating them with liquid ammonia
[53] or various amines [54,55]. Based on the crystal
structures, it was suggested that, during the crystalline
conversion from cellulose I to IIII, ammonia and amine
molecules penetrated between cellulose chains which were
originally connected to the intermolecular hydrogen bonds
in the cellulose I phase [56]. Therefore, it has been examined
as the ammonia fiber explosion (AFEX) pretreatment for
lingocellulosic biomass to improve the efficiency of
enzymatic conversion of it into sugars for the subsequent
production of biofuels and other products [57]. This was
interpreted as an increase in the cellulose IIII phase which
has a high reactivity toward enzymatic degradation [58].
Also, the soaking in aqueous ammonia (SAA) in solid-toliquid
ratio of 1:6 ~ 1:10 at 40 ~ 80ºC in a room atmosphere
has been used as the pretreatment of lignocellulose [59].
However, these processes have problems of requiring high
energy in keeping with the severe conditions and/or
recovering ammonia from aqueous solutions. Gaseous
ammonia is expected to be recovered with low energy.
Recently Kim et al. [60] developed a pretreatment using
gaseous ammonia, low-moisture anhydrous ammonia
(LMAA) pretreatment, where the lignocellulose was kept
in a flask filled with ammonia gas at 80ºC for 84 h.
We performed the LMAA pretreatment by modifying
the Kim method [25,26]. Water (100 g) was added dropwise
to the powdered dry Napier grass (100 g, volume 320 mL)
in a flask (1 L). The atmosphere of flask containing moist
powdered Napier grass was entirely replaced with gaseous
ammonia by evacuation with a pump under 20 mm Hg and
introduction of gaseous ammonia. The amount of ammonia
presented in the flask was 1.1 g which was smaller amount
compared with the SAA pretreatment. Moreover, the content
in flask did not involve the solution part, irrespective of the
solid-to-water ratio of 1:1. The moist powdered Napier
grass was kept under an ammonia gas atmosphere at room
temperature for 4 weeks. After the treatment, the ammonia
gas was removed with an evaporator. The treated Napier
grass was washed with water (2 L) repeatedly three times
and the brownish aqueous solution was separated. The
LMAA pretreatment did not induce the saccharification.
The effect of pretreatment was compared by the time
profile of CO2 evolution from SSF process of Napier grass
among LMAA-pretreatment, non-treatment, and alkalipretreatment
(Fig. 2). It is noteworthy that the SSF of
alkali-pretreated napiergrass was remarkably slowed down.
In the fermentation by S. cerevisiae of the alkali-pretreated
Napier grass, a nitrogen-source and a mineral were thought
to be insufficient, since the amino acids and the mineral
were removed from lignocelluloses by alkali-pretreatment
and the additional nutrients were not added in the SSF
process [61]. Moreover, the fermentation process might be
affected by the inhibitory materials derived from the alkalipretreatment
[60]. However, the LMAA-pretreatment did
not cause to the retardation of SSF rate.