Zhiwei Gong

Co-fermentation of cellobiose and xylose by Lipomyces starkeyi for lipid production.

Hydrolysates of lignocellulosic biomass contain glucose, xylose, arabinose, cellobiose, among other sugars. Effective utilization of these sugars remains challenging for microbial conversion, because most microorganisms consume such sugars sequentially with a strong preference for glucose. In the present study, the oleaginous yeast, Lipomyces starkeyi, was shown to consume cellobiose and xylose simultaneously and to produce intracellular lipids from cellobiose, xylose and glucose. In flask cultures with glucose, cellobiose or a mixture of cellobiose/xylose as carbon sources, overall substrate consumption rates were close to 0.6 g/L/h, and lipid coefficients were 0.19 g lipid/g sugar, respectively. This cellobiose/xylose co-fermentation strategy provides an opportunity to efficiently utilize lignocellulosic biomass for microbial lipid production, which is important for biorefinery and biofuel production.

Enzymatic hydrolysates of corn stover pretreated by a N-methylpyrrolidone–ionic liquid solution for microbial lipid production

In this study, we reported a novel N-methylpyrrolidone (NMP)–1-ethyl-3-methyl imidazolium acetate (EmimAc) mixed solvent (XILs = 0.2, mole fraction of ionic liquids (ILs) in the mixed solution) that can dissolve up to 10 wt% corn stover at 140 °C in 60 min. Physiochemical analysis showed major differences in terms of cellulose crystallinity, compositional distribution and surface morphology between raw corn stover samples and the regenerated materials, indicating that dissolution in the NMP–EmimAc system followed by regeneration with anti-solvents could be used as an effective pretreatment technology. Enzymatic saccharification of the regenerated corn stover afforded an 82.9% total reducing sugars yield and a 60.8% glucose yield within 24 h. The hydrolysates were used, without detoxification, as the carbon sources for the cultivation of Rhodosporidium Toruloides Y4 for lipid production. Glucose and xylose in the hydrolysates were both consumed in our process. In a word, our study reported a novel dissolution pretreatment technology for biomass utilization with outstanding advantages compared with traditional pure ionic liquid processes, such as (I) minimal use of expensive ILs in NMP, and commercially available EmimAc and NMP can be used directly without any further dry purification; (II) high cellulose and hemicellulose regeneration; (III) high enzymatic hydrolysis efficiency of the pretreated sample with full conversion of carbohydrates in less than 24 h; (IV) air-dried corn stover can be used directly (water content = ∼5%); (V) the hydrolysates can be used for further bioconversion and on inhibitory effect was observed. We believe that all of these advantages determine its potential for practical applications.

Kinetics of continuous cultivation of the oleaginous yeast Rhodosporidium toruloides

Microbial lipids are potential alternative feedstock for biofuel and oleochemical industries. The oleaginous yeast Rhodosporidium toruloides AS 2.1389 is an excellent lipid producer. To attain parameters for the understanding of the lipid production process, we performed continuous cultivation experiments under either carbon or nitrogen limitation. The maintenance coefficient and maximum cell mass yield for this yeast were determined as 5.7 mg glucose/g cell/h and 0.42 g cell/g glucose, respectively, under carbon limitation. Under nitrogen limitation, the highest lipid yield of 0.19 g/g was observed at the dilution rate of 0.02 h(-1) while the highest specific lipid formation rate of 0.058 g/g cell/h at the dilution rate of 0.08 h(-1). A kinetic model of lipid formation under steady state conditions was developed, parameters estimated, and optimal continuous cultivation conditions forecasted. These data should be very helpful to develop and design more efficient bioprocesses for microbial lipid production.

Lipid production from corn stover by the oleaginous yeast Cryptococcus curvatus

BackgroundMicrobial lipids produced from lignocellulosic biomass hold great promise for the biodiesel industry. These lipids usually consist of three major processes: pretreatment, enzymatic hydrolysis and lipid production. However, the conventional strategy of using biomass hydrolysates as the feedstock for lipid production suffers from low lipid coefficient and prohibitively high costs. More cost-effective and integrated processes are required to advance lignocellulosic biomass-based microbial lipid technology.ResultsThree different strategies were tested using the oleaginous yeast Cryptococcus curvatus ATCC 20509 as a lipid producer and alkaline-pretreated corn stover as a model material. It was found that the separate hydrolysis and enhanced lipid production process required more cellulolytic enzymes yet afforded a low lipid coefficient of 115.6 mg/g pretreated corn stover. When biomass hydrolysis and lipid production were integrated, the amounts of cellulase and xylanase were reduced and no β-glucosidase was required. The simultaneous saccharification and lipid production process gave a lipid coefficient of 129.4 mg/g pretreated corn stover. A higher lipid coefficient of 159.4 mg/g pretreated corn stover was obtained using the simultaneous saccharification and enhanced lipid production (SSELP) process. Furthermore, cellulolytic enzymes were found recoverable and reusable upon recycling the spent supernatants of the SSELP process, which could reduce enzyme consumption and wastewater discharge.ConclusionsThe SSELP process was superior to other processes in terms of converting alkaline-pretreated corn stover into lipids by C. curvatus, as it required less cellulolytic enzymes and had a higher lipid coefficient. Moreover, the process facilitated easy enzyme recycling that should lead to further reduction of enzyme consumption. These results provide valuable information for cost-effective lipid production from lignocelluloses, which should be particularly important in achieving a sustainable production of biodiesel.

Dynamics of the Lipid Droplet Proteome of the Oleaginous Yeast Rhodosporidium toruloides

ABSTRACT Lipid droplets (LDs) are ubiquitous organelles that serve as a neutral lipid reservoir and a hub for lipid metabolism. Manipulating LD formation, evolution, and mobilization in oleaginous species may lead to the production of fatty acid-derived biofuels and chemicals. However, key factors regulating LD dynamics remain poorly characterized. Here we purified the LDs and identified LD-associated proteins from cells of the lipid-producing yeast Rhodosporidium toruloides cultured under nutrient-rich, nitrogen-limited, and phosphorus-limited conditions. The LD proteome consisted of 226 proteins, many of which are involved in lipid metabolism and LD formation and evolution. Further analysis of our previous comparative transcriptome and proteome data sets indicated that the transcription level of 85 genes and protein abundance of 77 proteins changed under nutrient-limited conditions. Such changes were highly relevant to lipid accumulation and partially confirmed by reverse transcription-quantitative PCR. We demonstrated that the major LD structure protein Ldp1 is an LD marker protein being upregulated in lipid-rich cells. When overexpressed in Saccharomyces cerevisiae, Ldp1 localized on the LD surface and facilitated giant LD formation, suggesting that Ldp1 plays an important role in controlling LD dynamics. Our results significantly advance the understanding of the molecular basis of lipid overproduction and storage in oleaginous yeasts and will be valuable for the development of superior lipid producers.

Effects of selected ionic liquids on lipid production by the oleaginous yeast Rhodosporidium toruloides

Lignocellulosic biomass pretreatment with ionic liquids (ILs) has been emerged as a new technology, but the effects of residual ILs on the downstream biotransformation remain largely unknown. Here, three typical ILs were tested for their effects on lipid production by the oleaginous yeast Rhodosporidium toruloides AS 2.1389. When cultures were maintained at pH 6.0 in the presence of 30mM ILs, [Emim]Cl, [Emim][DEP], or [Emim][OAc], minor inhibition effects were observed. When cultures were performed in the presence of 60mM ILs or without pH control, inhibition was largely dependent on ILs. Detailed analysis indicated that the anion of [Emim][OAc] was assimilated, leading to a rapid alkaline-pH shift and enhanced inhibition on cell growth and lipid production. Our results demonstrated that R. toruloides is a robust lipid producer tolerating ILs at low concentrations, and that care should be taken in bioprocess control and data analysis when ILs are involved.

Simultaneous utilization of glucose and mannose from spent yeast cell mass for lipid production by Lipomyces starkeyi.

With ever-increasing culture of yeasts for the production of biofuels and other metabolites, spent yeast cell mass exceeds its traditional market demands. Yeast cell mass contains glucose, mannose and other sugars that may be utilized for microbial culture. Here we demonstrated that the oleaginous yeast Lipomyces starkeyi could utilize glucose and mannose simultaneously for lipid production. Overall substrate consumption rates and lipid coefficients were 0.58 g/L/h and 0.18 g lipid/g sugar, respectively, in flask cultures regardless of glucose, mannose or a mixture of both as the carbon source. L. starkeyi grew well on the hydrolysates of spent cell mass of Rhodosporidium toruloides, consumed both glucose and mannose therein, and produced lipid at a yield of 0.12 g lipid/g total reducing sugars. This co-utilization strategy expands carbon sources for lipid production. It should provide an opportunity for recycling spent cell mass and be of significant interests to biorefinery and biofuel production.

Recycling microbial lipid production wastes to cultivate oleaginous yeasts

To reduce wastes and the costs of microbial lipid production, it is imperative to recycle resources, including spent cell mass, mineral nutrients and water. In the present study, lipid production by the oleaginous yeast Rhodosporidium toruloides was used as a model system to demonstrate resources recycling. It was found that the hydrolysates of spent cell mass were good media to support cell growth of various oleaginous yeasts. When serial repitching experiments were performed using 70g/L glucose and the hydrolysates alone as nutrients, it produced 16.6, 14.6 and 12.9g/L lipids, for three successive cycles, while lipid titre remained almost constant when spent water was also recycled. The cell mass hydrolysates could be used as equivalents to the mixture of yeast extract and peptone to support lipid production from corn stalk hydrolysates. Our results showed efficient recycling of lipid production wastes and should be helpful to advance microbial lipid technology.

Co-fermentation of acetate and sugars facilitating microbial lipid production on acetate-rich biomass hydrolysates

The process of lignocellulosic biomass routinely produces a stream that contains sugars plus various amounts of acetic acid. As acetate is known to inhibit the culture of microorganisms including oleaginous yeasts, little attention has been paid to explore lipid production on mixtures of acetate and sugars. Here we demonstrated that the yeast Cryptococcus curvatus can effectively co-ferment acetate and sugars for lipid production. When mixtures of acetate and glucose were applied, C. curvatus consumed both substrates simultaneously. Similar phenomena were also observed for acetate and xylose mixtures, as well as acetate-rich corn stover hydrolysates. More interestingly, the replacement of sugar with equal amount of acetate as carbon source afforded higher lipid titre and lipid content. The lipid products had fatty acid compositional profiles similar to those of cocoa butter, suggesting their potential for high value-added fats and biodiesel production. This co-fermentation strategy should facilitate lipid production technology from lignocelluloses.

Co-utilization of corn stover hydrolysates and biodiesel-derived glycerol by Cryptococcus curvatus for lipid production.

In the present study, synergistic effects were observed when glycerol was co-fermented with glucose and xylose for lipid production by the oleaginous yeast Cryptococcus curvatus. Glycerol was assimilated simultaneously with sugars at the beginning of the culture without adaption time. Furthermore, better lipid production results, i.e., lipid yield and lipid productivity of 18.0g/100g and 0.13g/L/h, respectively, were achieved when cells were cultured in blends of corn stover hydrolysates and biodiesel-derived glycerol than those in the hydrolysates alone. The lipid samples had fatty acid compositional profiles similar to those of vegetable oils, suggesting their potential for biodiesel production. This co-utilization strategy provides an extremely simple solution to advance lipid production from both lignocelluloses and biodiesel-derived glycerol in one step.