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.

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.

"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.

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.

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.

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.

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.

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.

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.

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.

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.

Efficient conversion of sugarcane stalks into ethanol employing low temperature alkali pretreatment method.

An alternative route for bio-ethanol production from sugarcane stalks (juice and bagasse) featuring a previously reported low temperature alkali pretreatment method was evaluated. Test-tube scale pretreatment-saccharification experiments were carried out to determine optimal LTA pretreatment conditions for sugarcane bagasse with regard to the efficiency of enzymatic hydrolysis of the cellulose. Free fermentable sugars and bagasse recovered from 2 kg of sugarcane stalks were jointly converted into ethanol via separate enzymatic hydrolysis and fermentation (SHF). Results showed that 98% of the cellulose present in the optimally pretreated bagasse was hydrolyzed into glucose after 72-h enzymatic saccharification using commercially available cellulase and ß-glucosidase preparations at relatively low enzyme loading. The fermentable sugars in the mixture of the sugar juice and the bagasse hydrolysate were readily converted into 193.5 mL of ethanol by Saccharomyces cerevisiae within 12h, achieving 88% of the theoretical yield from the sugars and cellulose.

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.