Extracellular oil production by Rhodotorula paludigena BS15 for biorefinery without complex downstream processes.

To realize biomass refinery without complex downstream processes, we extensively screened for microbial strains that efficiently produce extracellular oil from sugars. Rhodotorula paludigena (formerly Rhodosporidium paludigenum) BS15 was found to efficiently produce polyol esters of fatty acids (PEFAs), which mainly comprised of 3-acetoxypalmitic acid and partially acetylated mannitol/arabinitol. To evaluate the performance of this strain, fed-batch fermentation was demonstrated on a flask scale, and 110 g/L PEFA and 103 g/L dry cells were produced in 12 days. To the best of our knowledge, the strain BS15 exhibited the highest PEFA titer (g/L) ever to be reported so far. Because the PEFA precipitated at the bottom of the culture broth, it could be easily recovered by simply discarding the upper phase. Various carbon sources can be utilized for cell growth and/or PEFA production, which signifies the potential for converting diverse biomass sources. Two different types of next-generation sequencers, Illumina HiSeq and Oxford Nanopore PromethION, were used to analyze the whole-genome sequence of the strain BS15. The integrative data analysis generated a high-quality and reliable reference genome for PEFA-producing R. paludigena. The 22.5-M base genome sequence and the estimated genes were registered in Genbank (accession numbers BQKY01000001-BQKY01000019). KEY POINTS: • R. paludigena BS15 was isolated after an extensive screening of extracellular oil producers from natural sources. • Fed-batch fermentation of R. paludigena BS15 yielded 110 g/L of PEFA, which is the highest titer ever reported to date. • Combined analysis using Illumina and Oxford Nanopore sequencers produced the near-complete genome sequence.

Controlled preparation of cellulases with xylanolytic enzymes from Trichoderma reesei (Hypocrea jecorina) by continuous-feed cultivation using soluble sugars.

The objective of this study was to develop an efficient production system for cellulase preparation with a high level of xylanolytic enzymes using soluble carbon sources. When xylose and arabinose were simultaneously fed with glucose and cellobiose, a mutant of Trichoderma reesei, M3-1, showed sufficient levels of cellulolytic and xylanolytic activities, indicating that xylose and arabinose are good inducers for the production of xylanolytic enzymes. In a continuous feeding experiment using glucose/cellobiose and glucose/xylose/cellobiose, cellulase preparations with various levels of xylanolytic enzymes were obtained by altering the feeding solutions and the timing of their addition. The volumetric production rates for xylanolytic activities at the glucose/xylose/cellobiose-feeding phase were significantly higher than at the glucose/cellobiose-feeding phase, while those for cellulolytic activities were comparable under the two conditions. Thus the composition of the enzyme preparation produced by the mutant was readily controlled by varying the inducers and the pattern of their addition, facilitating the tailored production of enzymes in a diversity of bioconversion processes.

Characterization of the Interactions between Cereal Flour and "Nata Puree" in Batter.

This study aimed to characterize the interactions between cereal flour (rice, wheat, and barley) and "nata puree" (NP), a disintegrated bacterial cellulose (BC) in the presence of a water-soluble polysaccharide, with powder-dispersion activity. Pasting properties of cereal flour with additives were analyzed using a Rapid Visco Analyzer, and disintegrated BC in water (BCW), three water-soluble polysaccharides: (1,3)(1,4)-ß-glucan, tamarind seed gum, and birchwood xylan, and the corresponding NPs were used as additives. For rice flour, additional BCW or NPs increased the initial and the peak viscosity. The addition of water-soluble polysaccharides produced the opposite trend: viscosity increased from the peak time to the end of measurements. For wheat flour, the addition of BCW or NP delayed the peak time and increased peak viscosity; the increase was maintained till the end of measurements. For barley flour, the additional BCW or NP caused a higher gelatinization rate and increased viscosity at the starch-retrogradation stage. Next, static gelatinization of a rice flour suspension in NP was successfully accomplished before placing it in a vessel; NP concentration in the gel significantly affected the firmness. Thus, the dynamic and unique interactions between various cereal flours and cell-wall polysaccharides in NPs can increase the flours' potential; static gelatinization of cereal flour with NP could expand flours' application range in both current and next-generation cooking.

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.

Evaluation of the Enzymatic Saccharification Efficiency of an Energy Crop, Erianthus arundinaceus, Pretreated with Ca(OH)2 Using both Countercurrent Washing System and pH Adjustment by Nonpressurized CO2.

Erianthus arundinaceus (ER) is greatly appreciated among domestic energy crops in Japan for the production of fermentable sugars from lignocellulosic polysaccharides. In this study, we developed an efficient Ca(OH)2-based pretreatment of both stems and leaves of ER at ambient temperature with the addition of a washing step for enzymatic saccharification. The recoveries of glucans and xylans in the pretreated ER after four countercurrent washing cycles were 91 and 76 %, respectively, the former being considerably higher than that of rice straw (RS) (72 %). Their saccharification ratios in the washed sample under the pressure of 1 atm CO2 were 80 and 92.5 %, respectively. The application of this simple sugar production process from ER would further support the domestic bioprocess development. ER is also foreseen to provide the additional feedstock favorable for harvesting from winter to spring in Japan, preventing a risk for feedstock shortage generated by single harvesting such as RS.

"Nata Puree," a Novel Food Material for Upgrading Vegetable Powders, Made by Bacterial Cellulose Gel Disintegration in the Presence of (1,3)(1,4)-ß-Glucan.

Pulverization is a potentially powerful solution for the resource management of surplus- and non-standard agricultural products, maintaining their nutritional values for long and ensuring their homogeneity, whereas their original textures could disappear to narrow the application ranges. Therefore, new technologies should be developed for reconstructing the powders to provide them with new physical characteristics. Herein, we developed a novel food material, nata puree (NP), by nata de coco (bacterial cellulose gel) disintegration with a water-soluble polysaccharide using a household blender. The process worked well with (1,3)(1,4)-ß-glucan (BGL) as the polysaccharide, which could be substituted with barley extract. Lichenase treatment of the NP dramatically modified its physical properties, suggesting the importance of the BGL polymeric forms. NP exhibited distinct potato powder and starch binding activities, which would be attributed to its interactions with the cell wall components and a physical capture of powders by the NP network, respectively. NP supplementation into the potato paste improved its firmness and enabled its printable range shift for 3D food printing to a lower powder-concentration. NP also promoted the dispersion of powders in its suspension, and designed gelation could also be successfully performed by the laser irradiation of an NP suspension containing dispersed curdlan and turmeric powders. Therefore, NP could be applied as a powder modifier to a wide range of products in both conventional cooking, food manufacturing, and next generation processes such as 3D food printing.

Crystallization and preliminary X-ray crystallographic studies of an exo-beta-D-glucosaminidase from Trichoderma reesei.

Chitosan is degraded to glucosamine (GlcN) by chitosanase and exo-beta-D-glucosaminidase (GlcNase). GlcNase from Trichoderma reesei (Gls93) is a 93 kDa extracellular protein composed of 892 amino acids. The enzyme liberates GlcN from the nonreducing end of the chitosan chain in an exo-type manner and belongs to glycoside hydrolase family 2. For crystallographic investigations, Gls93 was overexpressed in Pichia pastoris cells. The recombinant Gls93 had two molecular forms of approximately 105 kDa (Gls93-F1) and approximately 100 kDa (Gls93-F2), with the difference between them being caused by N-glycosylation. Both forms were crystallized by the hanging-drop vapour-diffusion method. Crystals of Gls93-F1 belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 98.27, b = 98.42, c = 108.28 A, and diffracted to 1.8 A resolution. Crystals of Gls93-F2 belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 67.84, b = 81.62, c = 183.14 A, and diffracted to 2.4 A resolution. Both crystal forms were suitable for X-ray structure analysis at high resolution.

Cellulase production on glucose-based media by the UV-irradiated mutants of Trichoderma reesei.

From 22,791 mutants of a cellulase hyper-producing strain of Trichoderma reesei (Hypocrea jecorina), ATCC66589, as the parent, we selected two mutants, M2-1 and M3-1, that produce cellulases in media containing both cellulose and glucose. The mutation enabled the mutants to produce cellulases, which were measured as p-nitrophenyl beta-D: -lactopyranoside-hydrolyzing activities, in media with glucose as a sole carbon source, although M2-1 exhibited different sensitivities to glucose from M3-1. When the mutants were grown for 8 days on a medium with cellulose as a sole carbon source, the filter-paper-degrading activities (FPAs) per gram of cellulose were 257 and 281 U for M2-1 and M3-1, respectively, values that were 1.1-1.2 times higher than that of the parental strain. Cellulase production by M2-1 and M3-1 on a medium with a continuously fed mixture of glucose and cellobiose resulted in 214 and 210 U of FPA/gram carbon sources, respectively, whereas less efficient production (140 U of FPA/gram carbon source) was achieved by the parental strain. The improved cellulase productivity of the mutants allows us to use glucose as a carbon source for efficient on-site production of cellulases with quality/quantity-controlled feeding of soluble carbon sources and inducers.

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.

Control of pH by CO 2 Pressurization for Enzymatic Saccharification of Ca(OH) 2 -Pretreated Rice Straw in the Presence of CaCO 3.

The aim of this study was to investigate the effect of pH control by CO 2 pressurization on the enzymatic hydrolysis of herbaceous feedstock in the calcium capturing by carbonation (CaCCO) process for fermentable sugar production. The pH of the slurry of 5 % (w/w) Ca(OH) 2 -pretreated/CO 2 -neutralized rice straw could be controlled between 5.70 and 6.38 at 50 °C by changing the CO 2 partial pressure ( p CO 2 ) from 0.1 to 1.0 MPa. A mixture of fungal enzyme preparations, namely, Trichoderma reesei cellulases/hemicellulases and Aspergillus niger ß-glucosidase, indicated that pH 5.5-6.0 is optimal for solubilizing sugars from Ca(OH) 2 -pretreated rice straw. Enzymatic saccharification of pretreated rice straw under various p CO 2 conditions revealed that the highest soluble sugar yields were obtained at p CO 2 0.4 MPa and over, which is consistent with the expected pH at the p CO 2 without enzymes and demonstrates the effectiveness of pH control by CO 2 pressurization.

The RURAL (reciprocal upgrading for recycling of ash and lignocellulosics) process: A simple conversion of agricultural resources to strategic primary products for the rural bioeconomy.

Rice straw (RS), an agricultural resource for lignocellulosic biorefineries, can deteriorate when sun-drying is ineffective. Poultry litter ash (PLA) has been considered as a renewable phosphorus source for crops but is highly alkaline. Here, a simple process was developed for their reciprocal upgrading. RS, PLA, and water were mixed for wet storage and alkali pretreatment of the RS at 25 °C for 14 d, and solid-solid separation was performed to obtain PLA-treated RS (PT-RS) and RS-treated PLA (RT-PLA). PT-RS was susceptible to enzymatic saccharification, and 65.5-68.6% of total sugar residues in PT-RS was converted to lactic acid by its nonsterile application for simultaneous saccharification and fermentation using Bacillus coagulans. RT-PLA exhibited 1.8-points lower pH and a more sensitive response of phosphorus solubilization to acid than those of PLA. This process could thus provide a breakthrough for the rural bioeconomy by manufacturing two strategic primary products for various commercial bioproducts.

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.

Washing Lime-Pretreated Rice Straw with Carbonated Water Facilitates Calcium Removal and Sugar Recovery in Subsequent Enzymatic Saccharification.

Generally, Ca(OH)2 pretreatment of lignocellulosics for fermentable sugar recovery requires a subsequent washing step for calcium removal and pH control for optimized saccharification. However, washing Ca(OH)2-pretreated feedstock with water is considered problematic because of the low solubility of Ca(OH)2 and its adsorption to biomass. In this study, we estimated the availability of carbonated water for calcium removal from the slurry of Ca(OH)2-pretreated rice straw (RS). We tested two kinds of countercurrent washing sequences, four washings exclusively with water (W4) and two washings with water and subsequent two washings with carbonated water (W2C2). The ratios of calcium removal from pretreatment slurry after washing were 64.2 % for the W4 process and 92.1 % for the W2C2 process. In the W2C2 process, 49 % of the initially added calcium was recovered as CaO by calcination. In enzymatic saccharification tests under a CO2 atmosphere at 1.5 atm, in terms of recovery of both glucose and xylose, pretreated, feedstock washed through the W2C2 process surpassed that washed through the W4 process, which could be attributed to the pH difference during saccharification: 5.6 in the W2C2 process versus 6.3 in the W4 process. Additionally, under an unpressurized CO2 atmosphere at 1 atm, the feedstock washed through the W2C2 process released 78.5 % of total glucose residues and 90.0 % of total xylose residues. Thus, efficient removal of calcium from pretreatment slurry would lead to not only the recovery of added calcium but also the proposal of a new, simple saccharification system to be used under an unpressurized CO2 atmosphere condition.

Reusable Floating Beads with Immobilized Xylose-Fermenting Yeast Cells for Simultaneous Saccharification and Fermentation of Lime-Pretreated Rice Straw.

Novel bioreactor beads for simultaneous saccharification and fermentation (SSF) of lime-pretreated rice straw (RS) into ethanol were prepared. Genetically modified Saccharomyces cerevisiae cells expressing genes encoding xylose reductase, xylitol dehydrogenase, and xylulokinase were immobilized in calcium alginate beads containing inorganic lightweight filler particles to reduce specific gravity. For SSF experiments, the beads were floated in slurry composed of lime-pretreated RS and enzymes and incubated under CO2 atmosphere to reduce the pH for saccharification and fermentation. Following this reaction, beads were readily picked up from the upper part of the slurry and were directly transferred to the next vessel with slurry. After 240 h of incubation, ethanol production by the beads was equivalent to that by free cells, a trend that was repeated in nine additional runs, with slightly improved ethanol yields. Slurry with pre-saccharified lime-pretreated RS was subjected to SSF with floating beads for 168 h. Although higher cell concentrations in beads resulted in more rapid initial ethanol production rates, with negligible diauxic behavior for glucose and xylose utilization, no improvement in the ethanol yield was observed. A fermentor-scale SSF experiment with floating beads was successfully performed twice, with repeated use of the beads, resulting in the production of 40.0 and 39.7 g/L ethanol. There was no decomposition of the beads during agitation at 60 rpm. Thus, this bioreactor enables reuse of yeast cells for efficient ethanol production by SSF of lignocellulosic feedstock, without the need for instruments for centrifugation or filtration of whole slurry.

Cellulase Production of Trichoderma reesei (Hypocrea jecorina) by Continuously Fed Cultivation Using Sucrose as Primary Carbon Source.

To expand the range of soluble carbon sources for our enzyme production system, we investigated the properties of sucrose utilization and its effect on cellulase production by Trichoderma reesei M2-1. We performed batch cultivation of T. reesei M2-1 on sucrose and related sugars along with cellobiose, which was used as a cellulase inducer. The results clearly revealed that the hydrolysis products of sucrose, i.e. glucose and fructose, but not sucrose, can be used as a carbon source for enzyme production. In a 10-day continuous feeding experiment using invertase-treated sucrose/cellobiose, the fungal strain produced cellulases with a filter paper-degrading activity of 20.3 U/mL and production efficiency of 254 U/g-carbon sources. These values were comparable with those of glucose/cellobiose feeding (21.2 U/mL and 265 U/g-carbon sources, respectively). Furthermore, the comparison of the specific activities clearly indicated that the compositions of both produced enzymes were similar. Therefore, enzymatically hydrolyzed sucrose can be utilized as an alternative carbon source to glucose in our enzyme production system with T. reesei M2-1.

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.

Candida utilis assimilates oligomeric sugars in rice straw hydrolysate via the Calcium-Capturing-by-Carbonation (CaCCO) process for glutathione- and cell-biomass production.

Rice-straw hydrolysate (RSH) prepared via the CaCCO (Calcium Capturing by Carbonation) process contains not only monosaccharides but also significant amounts of oligosaccharides. In this study, a glutathione-producing yeast, Candida utilis NBRC 0626, was found to assimilate those oligosaccharides. The yields of reduced glutathione (GSH) and dry cell weight (DCW) per consumed sugars in a medium with RSH after 72h incubation were 10.1mg/g-sugars and 0.49g/g-sugars, respectively. The yields were comparative to those in a medium containing a model monosaccharide mix, suggesting that the assimilated oligosaccharides contribute to additional GSH and DCW production. Glycosyl linkage analysis indicated that the yeast could cleave xylose-, galactose-, and arabinose residues as well as glucose residues at the non-reducing ends. After 72h incubation, 99.1% of the total glucose residues and 84.2% of the total xylose residues in RSH were depleted. Thus the yeast could be applied for efficient utilization of lignocellulosic hydrolysates.

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.