New Approach to Fuelization of Herbaceous Lignocelluloses through Simultaneous Saccharification and Fermentation Followed by Photocatalytic Reforming

Bio-fuelization of herbaceous lignocelluloses through a simultaneous saccharification and fermentation process (SSF) and photocatalytic reforming (photo-Reform) was examined. The SSF of the alkali-pretreated bamboo, rice straw, and silvergrass was performed in an acetate buffer (pH 5.0) using cellulase, xylanase, and Saccharomyces cerevisiae at 34 °C. Ethanol was produced in 63%–85% yields, while xylose was produced in 74%–97% yields without being fermented because xylose cannot be fermented by S. cerevisiae. After the removal of ethanol from the aqueous SSF solution, the SSF solution was subjected to a photo-Reform step where xylose was transformed into hydrogen by a photocatalytic reaction using Pt-loaded TiO2 (2 wt % of Pt content) under irradiation by a high pressure mercury lamp. The photo-Reform process produced hydrogen in nearly a yield of ten theoretical equivalents to xylose. Total energy was recovered as ethanol and hydrogen whose combustion energy was 73.4%–91.1% of that of the alkali-pretreated lignocelluloses (holocellulose). OPEN ACCESS Energies 2014, 7 4088


Introduction
Ethanol production from biomass has been receiving a great amount of interest from the viewpoint of being a renewable energy alternative to petroleum-based fuels [1].Second generation bioethanol production from lignocellulosic biomass has been recognized as one of the promising approaches, since the lignocelluloses are not directly in competition with food sources [2].Usual ethanol production from lignocellulose is conveniently achieved by simultaneous saccharification and fermentation (SSF) using Saccharomyces cerevisiae and hydrolytic enzymes [3,4].However, the ethanol yield is low compared with the first generation bioethanol produced from starches which are composed of glucose units (Equation ( 1)), because of the high content of hemicellulose composed of xylose units, which are not utilized by S. cerevisiae.Therefore fermentation of xylose has been performed using recombinant species of Escherichia coli [5][6][7][8] and S. cerevisiae [9][10][11][12].In order to develop a more convenient methodology to utilize xylose, we intend to develop photocatalytic reforming of xylose to hydrogen (Equation ( 2)) using a Pt-loaded titanium oxide (Pt-TiO 2 ) [13]:

C H O 2CO 2C H OH
The photocatalytic hydrogen evolution from H 2 O by the Pt-TiO 2 is initiated by the charge-separation on TiO 2 under photoexcitation [14].The electrons reduce water to generate H 2 on Pt while holes oxidize hydroxide to hydroxyl radical.It is well known that the use of electron-donating sacrificial agents remarkably accelerates TiO 2 -photocatalyzed hydrogen evolution since the hydroxyl radical is consumed by the sacrificial agents [15].Recently, we have found that sacrificial agents with all of the carbon attached oxygen atoms such as saccharides (e.g., glucose and xylose) and polyalcohols (e.g., l,2-ethanediol, glycerol, and arabitol) serve as an electron source until their sacrificial ability was exhausted in the TiO 2 -photocatalytic hydrogen evolution [16].Therefore, our attention has been focused on the photocatalytic reforming (photo-Reform) of biomasses using Pt-TiO 2 photocatalyst.Here we examined a new approach to fuelization of bamboo, rice straw, and silvergrass through SSF followed by photo-Reform (Scheme 1).Scheme 1. Conversion of lignocelluloses to hydrogen through SSF followed by photo-Reform.Operation: AL: alkali-pretreatment; SSF: simultaneous saccharification and fermentation; photo-Reform: photocatalytic reforming using a Pt-TiO 2 catalyst.  a) The SSF of the holocellulose was performed in a degassed acetate buffer solution (pH 5.0, 60 mL) at 34 °C using the cell suspension of S. cerevisiae (1.2 mL), cellulase (0.60 g), and xylanase (0.40 g).The amounts of holocellulose were set to 6.04 g, 3.90 g, and 3.75 g for bamboo, rice straw, and silvergrass respectively.Yields were based on the amounts of glucan and xylan in holocellulose; (b) Holocellulose was obtained by the pretreatment of lignocelluloses (50 g) with a 1% aqueous solution of NaOH (600 mL) at 95 °C for 1 h.The components of glucan and xylan were analyzed according to NREL method; (c) The weight of xylose was represented as W X .

Determination of Limiting Mole Amount of Hydrogen Evolved from Photo-Reform
Ethanol was recovered from the SSF solution by distillation under reduced pressure.The residual xylose in the SSF solution was subjected to the photo-Reform step.Pt-TiO 2 (100 mg, 1.25 mmol, 2 wt % of Pt) was introduced to the reaction vessel which was attached to the measuring cylinder.The SSF solution was added in reaction vessel so that the amounts of xylose became 0.25 (0.35), 0.50, 0.75, 1.00, and 1.25 mmol, and then the volume of the solution was adjusted to 150 mL by adding water.After the oxygen was purged from the suspension by bubbling it with N 2 gas, the irradiation was performed by a high-pressure mercury lamp under vigorous stirring with magnetic stirrer until the gas evolution ceased.Typical time conversion of the evolved gas is shown in Figure 1B.
Table 2 lists that the evolved gas volumes.The evolved gas increased with the increase of xylose.However, the molar ratios of H 2 and CO 2 to xylose (H 2 /xylose and CO 2 /xylose) were not proportional to the amount of xylose used.Therefore, the H 2 /xylose values were plotted against the molar ratio of xylose to catalyst (xylose/catalyst), as shown in Figure 2

Chemical Components of Lignocellulose
First, lignocelluloses were cut, dried, and made into a powder by a blender until the powder passed through a sieve with 150 μm mesh.The powdered lignocellulose (30 g) was treated with a 1% aqueous solution of NaOH (400 mL) at 95 °C for 1 h.The holocellulose was isolated as a pale yellow precipitate from the treated mixture by centrifugation at 10,000 rpm for 10 min and filtration.The supernatant solution was neutralized to pH 5.0 by a dilute HCl solution.The resulting dark brown precipitate, which was identified as lignin, was collected by centrifugation at 10,000 rpm for 10 min.Saccharides in the holocellulose were determined according to the methods published by the National Renewable Energy Laboratory (NREL) [19] as follows: sulfuric acid (72 wt %, 3.0 mL) was added slowly to holocellulose (300 mg) and kept at 30 °C for 1 h.The resulting solution was diluted by water (84 mL) until the concentration of sulfuric acid was 4 wt %.Acid hydrolysis was performed by autoclaving at 121 °C for 1 h.After the neutralization by CaCO 3 , the solution was subjected to a centrifugation to give the supernatant solution (ca.87 mL), which was concentrated to 30 mL by evaporation.The solution was analyzed by HPLC.The peaks of glucose and xylose appeared whereas the peaks of galactose and arabinose were very weak.The amounts of glucan and xylan were determined from the amounts of glucose and xylose determined by HPLC.It was confirmed that the total amounts of glucan and xylan were equal to the amounts of holocellolose.The ash component in lignocellulose was obtained by burning lignocellulose (2.0 g) in an electric furnace (KBF784N1, Koyo, Nara, Japan) for 2 h at 850 °C.Thus, the chemical components of lignocelluloses were determined, as shown in Table 1.

Procedures of SSF
The SSF was performed using the apparatus shown in Figure 5A.A cellulosic material and buffer solution (37.5 mL) were introduced in the reaction vessel and then autoclaved at 121 °C for 20 min.After cooling to room temperature under UV-irradiation, the hydrolytic enzyme dissolved in an acetate buffer solution (22.5 mL) and the cell suspension of S. cerevisiae were added to the suspension of the cellulosic material.The holocelluloses from the bamboo, rice straw, and silvergrass were set to 6.04 g, 3.90 g, and 3.75 g, respectively, which corresponded to the amounts of holocellulose in 10 g of the non-treated lignocellulose.After the air was purged with N 2 , the SSF was initiated by stirring the

Table 1 .
Production of ethanol and xylose through SSF of lignocelluloses.

Components (wt %) SSF process (a) Holocellulose (b) (glucan and xylan) Lignin Others Time (h) Ethanol (g) (yield (%)) Xylose (g) (c) (yield (%))
. As the xylose/catalyst values decreased, the H 2 /xylose values increased.The intercept of the plots represents H 2 max which is the limiting mole amount of H 2 obtained from one mole of xylose at an infinite amount of catalyst.The H 2 max values were nearly equal to the theoretical value (10.0) shown in Equation (2).The slopes of the plots were changed by the use of lignocellulose.It is possible that they would be affected by the amounts of the materials to lower catalytic activity.Also the limiting mole amount of CO 2 (CO 2 max ) were nearly equal to the theoretical values (5.0).Moreover, it was confirmed that the H 2 evolution from water was small (2 mL) in the absence of xylose.Other gases such as CH 4 and CO were not observed in the evolved gas.
(b)The v treated lignoce H 2 max and CO ication of the : W H = (2/150 e 2. Plots of ce straw; an le 2. Photo- transformed into hydrogen.The formed bio-fuel, ethanol and hydrogen, has almost the same combustion energy as the saccharide occurring in lignocelluloses.If the UV light in sunlight is used as the light source for catalytic reaction, this will provide a useful method to produce H 2 from biomass.