Evaluation of Two Sets of Sorghum Bagasse Samples as the Feedstock for Fermentable Sugar Recovery via the Calcium Capturing by Carbonation (CaCCO) Process.

Sorghum bagasse samples from two sets (n6 and bmr6; n18 and bmr18) of wild-type and corresponding "brown midrib" (bmr) mutant strains of sweet sorghum were evaluated as the feedstock for fermentable sugar recovery via the calcium capturing by carbonation (CaCCO) process, which involves Ca(OH)2 pretreatment of bagasse with subsequent neutralization with CO2 for enzymatic saccharification. Saccharification tests under various pretreatment conditions of the CaCCO process at different Ca(OH)2 concentrations, temperatures or residence periods indicated that bmr strains are more sensitive to the pretreatment than their counterparts are. It is expected that variant bmr6 is more suitable for glucose recovery than its wild-type counterpart because of the higher glucan content and better glucose recovery with less severe pretreatment. Meanwhile, bmr18showed higher scores of glucose recovery than its counterpart did, only at low pretreatment severity, and did not yield higher sugar recovery under the more severe conditions. The trend was similar to that of xylose recovery data from the two bmr strains. The advantages of bmr strains were also proven by means of simultaneous saccharification and fermentation of CaCCO-pretreated bagasse samples by pentose-fermenting yeast strain Candida shehatae Cs 4R. The amounts needed for production of 1 L of ethanol from n6, bmr6, n18, and bmr18samples were estimated as 4.11, 3.46, 4.03, and 3.95 kg, respectively. The bmr strains seem to have excellent compatibility with the CaCCO process for ethanol production, and it is expected that integrated research from the feedstock to bioprocess may result in breakthroughs for commercialization.

Integration of a phenolic-acid recovery step in the CaCCO process for efficient fermentable-sugar recovery from rice straw.

An advanced sugar-platform bioprocess for lignocellulosic feedstocks by adding a phenolic-acid (PA: p-coumaric acid and ferulic acid) recovery step to the CaCCO process was designed. For efficient PA extraction, pretreatment was 95°C for 2h, producing a yield of 7.30 g/kg-dry rice straw (65.2% of total ester-linked PAs) with insignificant effects on saccharification. PAs were readily recovered in solution during the repeated washings of solids, and the glucose yield, after 72-h saccharification of the washed solids, was significantly improved from 65.9% to 70.3-72.7%, suggesting the removal of potential enzyme inhibitors. The promotion of xylose yield was insignificant, probably due to 13.1-17.8% loss of xylose residues after washing(s). This new bioprocess, termed the SRB (simultaneous recovery of by-products)-CaCCO process, would effectively produce fermentable sugars and other valuables from feedstocks, strengthening the platform in both economic and environmental terms.

Expression of neutral ß-glucosidase from Scytalidium thermophilum in Candida glabrata for ethanol production from alkaline-pretreated rice straw.

We successfully expressed the neutral ß-glucosidase (BGL4) from Scytalidium thermophilum in the thermotolerant yeast Candida glabrata. Compared to the strain expressing Aspergillus acidic ß-glucosidase (BGL1), the BGL4-expressing strain showed a higher cellobiose fermentation ability at pH 6.0 and 40°C, leading to a higher ethanol production from alkaline-pretreated rice straw.

Sequential incubation of Candida shehatae and ethanol-tolerant yeast cells for efficient ethanol production from a mixture of glucose, xylose and cellobiose.

A mixture of 5% (w/v) glucose, 4% (w/v) xylose and 5% (w/v) cellobiose was fermented into ethanol using non-recombinant yeasts. Two series of experiments were carried out: (1) sequential fermentation with Candida shehatae D45-6 and Saccharomyces cerevisiae (Cs-Sc), and (2) sequential fermentation with C. shehatae D45-6 and Brettanomyces bruxellensis (Cs-Bb). C. shehatae D45-6 was initially used for glucose and xylose fermentation before adding highly ethanol-tolerant yeasts, either S. cerevisiae or B. bruxellensis, for cellobiose fermentation. For the sequential fermentation using S. cerevisiae, ß-glucosidase was also included in the second step. In these two experiments, ethanol concentration reached 5.6-5.8% (w/v) and 99% sugar was consumed. Our results suggest that restricted glucose production from cellulose by saccharification could allow D45-6 to complete monosaccharide fermentation before the ethanol concentration exceeded its tolerance level.

Ethanol production by repeated-batch simultaneous saccharification and fermentation (SSF) of alkali-treated rice straw using immobilized Saccharomyces cerevisiae cells.

Repeated-batch simultaneous saccharification and fermentation (SSF) of alkali-treated rice straw using immobilized yeast was developed to produce ethanol. Saccharomyces cerevisiae cells were immobilized by entrapping in photocrosslinkable resin beads, and we evaluated the possibility of its reuse and ethanol production ability. In batch SSF of 20% (w/w) rice straw, the ethanol yields based on the glucan content of the immobilized cells were slightly low (76.9% of the theoretical yield) compared to free cells (85.2% of the theoretical yield). In repeated-batch SSF of 20% (w/w) rice straw, stable ethanol production of approx. 38gL(-1) and an ethanol yield of 84.7% were obtained. The immobilizing carrier could be reused without disintegration or any negative effect on ethanol production ability.

Readily-milled fraction of wet sugarcane bagasse as an advanced feedstock for monosaccharide production via the RT-CaCCO process.

The RT-CaCCO process for enzymatic saccharification was applied to readily-milled fractions of wet sugarcane bagasse. Wet bagasse immediately after juice extraction was crushed with shark-mill blades to prepare two fractions referred to as readily-milled (RF) and hardly-milled fraction (HF). Monosaccharide recoveries from RFs via the RT-CaCCO process were 1.03-1.21 times higher than those from HFs. Moreover, when the wet weight ratio of RF/HF was adjusted to 2/8, the hexose recovery from RF was 90.9%, which was 1.3 times higher than that of the wet bagasse before fractionation. The results show that this process can be used for efficient monosaccharide recovery from RF of wet bagasse. In addition, the process can be adapted to more fibrous HF for multiple uses such as fuel for boilers and fibers for particleboards.

Improved ethanol and reduced xylitol production from glucose and xylose mixtures by the mutant strain of Candida shehatae ATCC 22984.

A mutant Cs3512, which showed better fermentation of xylose and the mixtures of xylose and glucose, was obtained through mutation of Candida shehatae ATCC 22984 and screening with a medium containing antimycin A and TTC (2,3,5-triphenyltetrazolium chloride). Cs3512 produced 44.4 g/l of ethanol from 121.3 g/l of xylose, which was 13% higher than that by ATCC 22984. At the same time, xylitol production was reduced by 38% to 10.2 g/l from 16.3 g/l by ATCC 22984. Cs3512 also showed 8% increase in ethanol yield from 0.39 to 0.42 g/g comparing to ATCC 22984 when fermenting the sugar mixture composed of 52.9 g/l glucose and 21.2 g/l xylose. When Cs3512 was used in the simultaneous saccharification and fermentation of lime pretreated rice straw via CaCCO (calcium capturing by carbonation) process, it produced ethanol at 77% of the theoretical yield. The results imply that Cs3512 is a potential non-recombinant yeast strain for ethanol production from lignocellulosic biomass.

DiSC (direct saccharification of culms) process for bioethanol production from rice straw.

A simple process (the direct-saccharification-of-culms (DiSC) process) to produce ethanol from rice straw culms, accumulating significant amounts of soft carbohydrates (SCs: glucose, fructose, sucrose, starch and ß-1,3-1,4-glucan) was developed. This study focused on fully mature culms of cv. Leafstar, containing 69.2% (w/w of dried culms) hexoses from SCs and cellulose. Commercially-available wind-separation equipment successfully prepared a culm-rich fraction with a SC recovery of 83.1% (w/w) from rice straw flakes (54.1% of total weight of rice straw). The fraction was suspended in water (20%, w/w) for starch liquefaction, and the suspension was subjected to a simultaneous saccharification and fermentation with yeast, yielding 5.6% (w/v) ethanol (86% of the theoretical yield from whole hexoses in the fraction) after 24h fermentation. Thus, the DiSC process produced highly-concentrated ethanol from rice straw in a one vat process without any harsh thermo-chemical pretreatments.

An improved CARV process for bioethanol production from a mixture of sugar beet mash and potato mash.

A mixed mash of sugar beet roots and potato tubers with a sugar concentration of 23.7% w/v was used as a feedstock for bioethanol production. Enzymatic digestion successfully reduced the viscosity of the mixture, enabling subsequent heat pretreatment for liquefaction/sterilization. An energy-consuming thick juice preparation from sugar beet for concentration and sterilization was omitted in this new process.

Bioethanol production from rice straw by a sequential use of Saccharomyces cerevisiae and Pichia stipitis with heat inactivation of Saccharomyces cerevisiae cells prior to xylose fermentation.

In order to establish an efficient bioethanol production system from rice straw, a new strategy to ferment the mixture of glucose and xylose by a sequential application of Saccharomyces cerevisiae and Pichia stipitis was developed, in which heat inactivation of S. cerevisiae cells before addition of P. stipitis was employed. The results showed that heating at 50°C for 6h was sufficient to give high xylose fermentation efficiency. By application of the inactivation process, 85% of the theoretical yield was achieved in the fermentation of the synthetic medium. At the same time, the xylitol production was reduced by 42.4% of the control process. In the simultaneous saccharification and fermentation of the lime-pretreated and CO(2)-neutralized rice straw, the inactivation of S. cerevisiae cells enabled the full conversion of glucose and xylose within 80 h. Finally, 21.1g/l of ethanol was produced from 10% (w/w) of pretreated rice straw and the ethanol yield of rice straw reached 72.5% of the theoretical yield. This process is expected to be useful for the ethanol production from lignocellulosic materials in the regions where large-scale application of recombinant microorganisms was restricted.

RT-CaCCO process: an improved CaCCO process for rice straw by its incorporation with a step of lime pretreatment at room temperature.

We improved the CaCCO process for rice straw by its incorporation with a step of lime pretreatment at room temperature (RT). We firstly optimized the RT-lime pretreatment for the lignocellulosic part. When the ratio of lime/dry-biomass was 0.2 (w/w), the RT lime-pretreatment for 7-d resulted in an effect on the enzymatic saccharification of cellulose and xylan equivalent to that of the pretreatment at 120°C for 1h. Sucrose, starch and ß-1,3-1,4-glucan, which could be often detected in rice straw, were mostly stable under the RT-lime pretreatment condition. Then, the pretreatment condition in the conventional CaCCO process was modified by the adaptation of the optimized RT lime-pretreatment, resulting in significantly better carbohydrate recoveries via enzymatic saccharification than those of the CaCCO process (120°C for 1 h). Thus, the improved CaCCO process (the RT-CaCCO process) could preserve/pretreat the feedstock at RT in a wet form with minimum loss of carbohydrates.

Characterization of starch granules in rice culms for application of rice straw as a feedstock for saccharification.

Rice plants are known to accumulate starch in leaf sheaths and culms, and in some cultivars significant amounts of starch are present at the mature stage. This can be considered as potential feedstock for the recovery of fermentable sugars. We isolated starches from the culms of cultivars Yumeaoba, Koshihikari, and Leafstar to investigate their structural and physical features. Yumeaoba culm starch contained 20.2% amylose, whereas Koshihikari and Leafstar contained 25.8% and 25.2%. Yumeaoba culm starch was found by chain-length distribution analysis to contain higher amounts of short chains, resulting in lower gelatinization temperature by 7 degrees C, as compared to Koshihikari and Leafstar. Consequently, the rate of enzymatic hydrolysis of Yumeaoba culm starches reached maximum at a lower temperature than Leafstar. Rice culm starch, with a lower gelatinization temperature, can provide an advantageous material for feedstock for bioethanol production in terms of energy conservation.

A novel lime pretreatment for subsequent bioethanol production from rice straw--calcium capturing by carbonation (CaCCO) process.

In order to establish an efficient bioethanol production system for rice straw, we developed a novel lime-pretreatment process (CaCCO process) that did not require a solid-liquid-separation step. This process adopted a step in which after pretreatment lime was neutralized by carbonation, resulting in a final pH of about 6. CaCO(3) produced by the process was kept in the reaction vessel, and no significant inhibitory effects on enzymatic saccharification and fermentation were observed. In the CaCCO process, solubilized carbohydrates, such as xylan, starch, and sucrose were also kept in the vessel, enabling high recoveries of monomeric sugars. Simultaneous saccharification and fermentation (SSF) of pretreated rice straw, 10% (g-rice straw/g-water), using Saccharomyces cerevisiae and Pichia stipitis yielded 19.1 g L(-1) ethanol that was 74% of the theoretical yield from glucose and xylose. Thus, this process represents a novel pretreatment method to utilize not only cellulose but also xylan, starch, and sucrose from biomass.

Culm in rice straw as a new source for sugar recovery via enzymatic saccharification.

Rice straw was manually dissected and two main fractions were recovered: a culm and a leaf sheath/blade fraction, in order to evaluate their potential as feedstocks for the recovery of fermentable sugars. In the case of cv. Koshihikari and Milkyqueen, most soft carbohydrates (SCs: glucose, fructose, sucrose, starch, and beta-1,3-1,4-glucan) were present in the culms, reaching 47.9% and 89.2% of total SCs in the two main fractions. The results also indicated that beta-glucans (cellulose and beta-1,3-1,4-glucan) and xylan in the culms were more susceptible to direct enzymatic attack than those in the leaf sheath/blades. Thus the culm has high potential as a new feedstock for the extraction of fermentable sugars in a concentrated form, as compared to whole rice straw and the leaf sheath/blade. In this study, a novel method of separating a culm from the whole rice straw by means of wind power was also evaluated.

Efficient recovery of glucose and fructose via enzymatic saccharification of rice straw with soft carbohydrates.

Soft carbohydrates, defined as readily-recoverable carbohydrates via mere extraction from the biomass or brief enzymatic saccharification, were found in significant amounts in rice straw as forms of free glucose, free fructose, sucrose, starch, and beta-1,3-1,4-glucan. In this study, we investigated their amounts in rice straw (defined as culm and leaf sheath), and developed an easy method for glucose and fructose recovery from them with heat-pretreatment and subsequent 4-h enzymatic saccharification with an enzyme cocktail of cellulase and amyloglucosidase. The recovery of glucose and fructose exhibited good correlation with the amounts of soft carbohydrates. The maximum yields of glucose and fructose in the rice straw per dry weight at the heading stage and the mature stage were 43.5% in cv. Habataki and 34.1% in cv. Leafstar. Thus, rice straw with soft carbohydrates can be regarded as a novel feedstock for economically feasible production of readily-fermentable glucose and fructose for bioethanol.