Improved bioconversion of lignocellulosic biomass by Saccharomyces cerevisiae engineered for tolerance to acetic acid

Lignocellulosic biomass has considerable potential for the production of fuels and chemicals as a promising alternative to conventional fossil fuels. However, the bioconversion of lignocellulosic biomass to desired products must be improved to reach economic viability. One of the main technical hurdles is the presence of inhibitors in biomass hydrolysates, which hampers the bioconversion efficiency by biorefinery microbial platforms such as Saccharomyces cerevisiae in terms of both production yields and rates. In particular, acetic acid, a major inhibitor derived from lignocellulosic biomass, severely restrains the performance of engineered xylose‐utilizing S. cerevisiae strains, resulting in decreased cell growth, xylose utilization rate, and product yield. In this study, the robustness of XUSE, one of the best xylose‐utilizing strains, was improved for the efficient conversion of lignocellulosic biomass into bioethanol under the inhibitory condition of acetic acid stress. Through adaptive laboratory evolution, we successfully developed the evolved strain XUSAE57, which efficiently converted xylose to ethanol with high yields of 0.43–0.50 g ethanol/g xylose even under 2–5 g/L of acetic stress. XUSAE57 not only achieved twofold higher ethanol yields but also improved the xylose utilization rate by more than twofold compared to those of XUSE in the presence of 4 g/L of acetic acid. During fermentation of lignocellulosic hydrolysate, XUSAE57 simultaneously converted glucose and xylose with the highest ethanol yield reported to date (0.49 g ethanol/g sugars). This study demonstrates that the bioconversion of lignocellulosic biomass by an engineered strain could be significantly improved through adaptive laboratory evolution for acetate tolerance, which could help realize the development of an economically feasible lignocellulosic biorefinery to produce fuels and chemicals.     

Co‐hydrolysis of lignocellulosic biomass for microbial lipid accumulation

The herbaceous perennial energy crops miscanthus, giant reed, and switchgrass, along with the annual crop residue corn stover, were evaluated for their bioconversion potential. A co‐hydrolysis process, which applied dilute acid pretreatment, directly followed by enzymatic saccharification without detoxification and liquid–solid separation between these two steps was implemented to convert lignocellulose into monomeric sugars (glucose and xylose). A factorial experiment in a randomized block design was employed to optimize the co‐hydrolysis process. Under the optimal reaction conditions, corn stover exhibited the greatest total sugar yield (glucose + xylose) at 0.545 g g^?1 dry biomass at 83.3% of the theoretical yield, followed by switch grass (0.44 g g^?1 dry biomass, 65.8% of theoretical yield), giant reed (0.355 g g^?1 dry biomass, 64.7% of theoretical yield), and miscanthus (0.349 g g^?1 dry biomass, 58.1% of theoretical yield). The influence of combined severity factor on the susceptibility of pretreated substrates to enzymatic hydrolysis was clearly discernible, showing that co‐hydrolysis is a technically feasible approach to release sugars from lignocellulosic biomass. The oleaginous fungus Mortierella isabellina was selected and applied to the co‐hydrolysate mediums to accumulate fungal lipids due to its capability of utilizing both C5 and C6 sugars. Fungal cultivations grown on the co‐hydrolysates exhibited comparable cell mass and lipid production to the synthetic medium with pure glucose and xylose. These results elucidated that combining fungal fermentation and co‐hydrolysis to accumulate lipids could have the potential to enhance the utilization efficiency of lignocellulosic biomass for advanced biofuels production. Biotechnol. Bioeng. 2013; 110: 1039–1049. © 2012 Wiley Periodicals, Inc.     

Combined Cell Surface Display of β‐d‐Glucosidase (BGL), Maltose Transporter (MAL11), and Overexpression of Cytosolic Xylose Reductase (XR) in Saccharomyces cerevisiae Enhance Cellobiose/Xylose Coutilization for Xylitol Bioproduction from Lignocellulosic Biomass

Xylitol is a highly valuable commodity chemical used extensively in the food and pharmaceutical industries. The production of xylitol from d ‐xylose involves a costly and polluting catalytic hydrogenation process. Biotechnological production from lignocellulosic biomass by micro‐organisms like yeasts is a promising option. In this study, xylitol is produced from lignocellulosic biomass by a recombinant strain of Saccharomyces cerevisiae (S. cerevisiae) (YPH499‐SsXR‐AaBGL) expressing cytosolic xylose reductase (Scheffersomyces stipitis xylose reductase [SsXR]), along with a β‐d ‐glucosidase (Aspergillus aculeatus β‐glucosidase 1 [AaBGL]) displayed on the cell surface. The simultaneous cofermentation of cellobiose/xylose by this strain leads to an ≈2.5‐fold increase in Yxylitol/xylose (=0.54) compared to the use of a glucose/xylose mixture as a substrate. Further improvement in the xylose uptake by the cell is achieved by a broad evaluation of several homologous and heterologous transporters. Homologous maltose transporter (ScMAL11) shows the best performance in xylose transport and is used to generate the strain YPH499‐XR‐ScMAL11‐BGL with a significantly improved xylitol production capacity from cellobiose/xylose coutilization. This report constitutes a promising proof of concept to further scale up the biorefinery industrial production of xylitol from lignocellulose by combining cell surface and metabolic engineering in S. cerevisiae.     

d‐lactic acid production from renewable lignocellulosic biomass via genetically modified Lactobacillus plantarum

d ‐lactic acid is of great interest because of increasing demand for biobased poly‐lactic acid (PLA). Blending poly‐l ‐lactic acid with poly‐d ‐lactic acid greatly improves PLA's mechanical and physical properties. Corn stover and sorghum stalks treated with 1% sodium hydroxide were investigated as possible substrates for d ‐lactic acid production by both sequential saccharification and fermentation and simultaneous saccharification and cofermentation (SSCF). A commercial cellulase (Cellic CTec2) was used for hydrolysis of lignocellulosic biomass and an l ‐lactate‐deficient mutant strain Lactobacillus plantarum NCIMB 8826 ldhL1 and its derivative harboring a xylose assimilation plasmid (ΔldhL1‐pCU‐PxylAB) were used for fermentation. The SSCF process demonstrated the advantage of avoiding feedback inhibition of released sugars from lignocellulosic biomass, thus significantly improving d ‐lactic acid yield and productivity. d ‐lactic acid (27.3 g L^?1) and productivity (0.75 g L^?1 h^?1) was obtained from corn stover and d ‐lactic acid (22.0 g L^?1) and productivity (0.65 g L^?1 h^?1) was obtained from sorghum stalks using ΔldhL1‐pCU‐PxylAB via the SSCF process. The recombinant strain produced a higher concentration of d ‐lactic acid than the mutant strain by using the xylose present in lignocellulosic biomass. Our findings demonstrate the potential of using renewable lignocellulosic biomass as an alternative to conventional feedstocks with metabolically engineered lactic acid bacteria to produce d ‐lactic acid. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:271–278, 2016     

Enabling xylose utilization in Yarrowia lipolytica for lipid production

The conversion of lignocellulosic sugars, in particular xylose, is important for sustainable fuels and chemicals production. While the oleaginous yeast Yarrowia lipolytica is a strong candidate for lipid production, it is currently unable to effectively utilize xylose. By introducing a heterologous oxidoreductase pathway and enabling starvation adaptation, we obtained a Y. lipolytica strain, E26 XUS, that can use xylose as a sole carbon source and produce over 15 g/L of lipid in bioreactor fermentations (29.3% of theoretical yield) with a maximal lipid productivity of 0.19 g/L/h. Genomic sequencing and genetic analysis pointed toward increases in genomic copy number of the pathway and resulting elevated expression levels as the causative mutations underlying this improved phenotype. More broadly, many regions of the genome were duplicated during starvation of Yarrowia. This strain can form the basis for further engineering to enhance xylose catabolic rates and conversion. Finally, this study also reveals the flexibility and dynamic nature of the Y. lipolytica genome, and the means at which starvation can be used to induce genomic duplications.     

Producing Biochemicals in Yarrowia lipolytica from Xylose through a Strain Mating Approach

Enabling xylose catabolism is challenging, especially for unconventional yeasts and previously engineered background strains. In this study, the efficacy of a yeast mating approach with Yarrowia lipolytica that can combine a previously engineering and evolved xylose phenotype with a metabolite overproduction phenotype is demonstrated. Specifically, several engineered Y. lipolytica strains that produce α‐linolenic acid (ALA), riboflavin, and triacetic acid lactone (TAL) with an engineered and adapted xylose‐utilizing strain to obtain three diploid strains that rapidly produce these molecules directly from xylose are mated. Titers of 0.52 g L^?1 ALA, 96.6 mg L^?1 riboflavin, and 2.9 g L^?1 TAL, are obtained from xylose in flask cultures and 1.42 g L^?1 production of ALA is obtained using bioreactor condition. This total production level is generally on par or higher than the parental strain cultivated on glucose, although specific productivities decreased as a result of improved overall cell growth by the diploid strains. In the case of ALA, this lipid content reached similar levels to that of flaxseed oil. This result showcases the first study using strain mating in Y. lipolytica for producing biomolecules from xylose, and thus demonstrates the utility of this approach as a routine tool for metabolic engineering.     

Engineering of Klebsiella oxytoca for production of 2,3‐butanediol using mixed sugars derived from lignocellulosic hydrolysates

2,3‐Butanediol (2,3‐BDO) is a promising bulk chemical owing to its high potential in industrial applications. Here, we engineered Klebsiella oxytoca for the economic production of 2,3‐BDO using mixed sugars from renewable biomass. First, to improve xylose consumption, the xylose transporter gene (xylE) was integrated into the methylglyoxal synthase A (mgsA)‐coding gene loci, and the engineered CHA004 strain showed much faster consumption of xylose than wild‐type (WT) strain with 1.4‐fold increase of overall sugar consumption rate. To further improve sugar utilization, we performed adaptive laboratory evolution for 90 days. The evolved strain (CHA006) was evaluated by cultivating it in the media containing single‐ or mixed‐sugars, and it was clearly observed that CHA006 has improved sugar consumption and 2,3‐BDO production than those of the parental strain. Finally, we demonstrated the superiority of CHA006 by culturing in two lignocellulosic hydrolysates derived from sunflower or pine tree. Particularly, in the pine tree hydrolysate containing xylose, glucose, galactose, and mannose, the CHA006 strain showed much improved consumption rates for all sugars, and 2,3‐BDO productivity (0.73 g L^?1 hr^?1) increased by 3.2‐fold compared to WT strain. We believe that the engineered CHA006 strain can be a potential host in the development of economic bioprocess for 2,3‐BDO through efficient utilization of mixed sugars derived from lignocellulosic biomass.     

Metabolic engineering of Yarrowia lipolytica to produce chemicals and fuels from xylose

Yarrowia lipolytica is a biotechnological chassis for the production of a range of products, such as microbial oils and organic acids. However, it is unable to consume xylose, the major pentose in lignocellulosic hydrolysates, which are considered a preferred carbon source for bioprocesses due to their low cost, wide abundance and high sugar content.Here, we engineered Y. lipolytica to metabolize xylose to produce lipids or citric acid. The overexpression of xylose reductase and xylitol dehydrogenase from Scheffersomyces stipitis were necessary but not sufficient to permit growth. The additional overexpression of the endogenous xylulokinase enabled identical growth as the wild type strain in glucose. This mutant was able to produce up to 80 g/L of citric acid from xylose. Transferring these modifications to a lipid-overproducing strain boosted the production of lipids from xylose. This is the first step towards a consolidated bioprocess to produce chemicals and fuels from lignocellulosic materials.     

Biotechnological conversions of bio‐diesel‐derived crude glycerol by Yarrowia lipolytica strains

In the present report, crude glycerol, waste discharged from bio‐diesel production, was used as carbon substrate for three natural Yarrowia lipolytica strains (LFMB 19, LFMB 20 and ACA‐YC 5033) during growth in nitrogen‐limited submerged shake‐flask experiments. In media with initial glycerol concentration of 30 g/L, all strains presented satisfactory microbial growth and complete glycerol uptake. Although culture conditions favored the secretion of citric acid (and potentially the accumulation of storage lipid), for the strains LFMB 19 and LFMB 20, polyol mannitol was the principal metabolic product synthesized (maximum quantity 6.0 g/L, yield 0.20–0.26 g per g of glycerol consumed). The above strains produced small quantities of lipids and citric acid. In contrast, Y. lipolytica ACA‐YC 5033 produced simultaneously higher quantities of lipid and citric acid and was further grown on crude glycerol in nitrogen‐limited experiments, with constant nitrogen and increasing glycerol concentrations (70–120 g/L). Citric acid and lipid concentrations increased with increment of glycerol; maximum total citric acid 50.1 g/L was produced (yield 0.44 g per g of glycerol) while simultaneously 2.0 g/L of fat were accumulated inside the cells (0.31 g of lipid per g of dry weight). Cellular lipids were mainly composed of neutral fraction, the concentration of which substantially increased with time. Moreover, in any case, the phospholipid fraction was more unsaturated compared with total and neutral lipids, while at the early growth step, microbial lipid was more rich in saturated fatty acids (e.g. C16:0 and C18:0) compared with the stationary phase.     

Production and characterization of poly‐3‐hydroxybutyrate from biodiesel‐glycerol by Burkholderia cepacia ATCC 17759

Glycerol, a byproduct of the biodiesel industry, can be used by bacteria as an inexpensive carbon source for the production of value‐added biodegradable polyhydroxyalkanoates (PHAs). Burkholderia cepacia ATCC 17759 synthesized poly‐3‐hydroxybutyrate (PHB) from glycerol concentrations ranging from 3% to 9% (v/v). Increasing the glycerol concentration results in a gradual reduction of biomass, PHA yield, and molecular mass (M_n and M_w) of PHB. The molecular mass of PHB produced utilizing xylose as a carbon source is also decreased by the addition of glycerol as a secondary carbon source dependent on the time and concentration of the addition. ¹H‐NMR revealed that molecular masses decreased due to the esterification of glycerol with PHB resulting in chain termination (end‐capping). However, melting temperature and glass transition temperature of the end‐capped polymers showed no significant difference when compared to the xylose‐based PHB. The fermentation was successfully scaled up to 200 L for PHB production and the yield of dry biomass and PHB were 23.6 g/L and 7.4 g/L, respectively. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Xylitol does not inhibit xylose fermentation by engineered <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> expressing <Emphasis Type="Italic">xylA</Emphasis> as severely as it inhibits xylose isomerase reaction in vitro

Efficient fermentation of xylose, which is abundant in hydrolysates of lignocellulosic biomass, is essential for producing cellulosic biofuels economically. While heterologous expression of xylose isomerase in Saccharomyces cerevisiae has been proposed as a strategy to engineer this yeast for xylose fermentation, only a few xylose isomerase genes from fungi and bacteria have been functionally expressed in S. cerevisiae. We cloned two bacterial xylose isomerase genes from anaerobic bacteria (Bacteroides stercoris HJ-15 and Bifidobacterium longum MG1) and introduced them into S. cerevisiae. While the transformant with xylA from B. longum could not assimilate xylose, the transformant with xylA from B. stercoris was able to grow on xylose. This result suggests that the xylose isomerase (BsXI) from B. stercoris is functionally expressed in S. cerevisiae. The engineered S. cerevisiae strain with BsXI consumed xylose and produced ethanol with a good yield (0.31 g/g) under anaerobic conditions. Interestingly, significant amounts of xylitol (0.23 g xylitol/g xylose) were still accumulated during xylose fermentation even though the introduced BsXI might not cause redox imbalance. We investigated the potential inhibitory effects of the accumulated xylitol on xylose fermentation. Although xylitol inhibited in vitro BsXI activity significantly (K _I = 5.1 ± 1.15 mM), only small decreases (less than 10%) in xylose consumption and ethanol production rates were observed when xylitol was added into the fermentation medium. These results suggest that xylitol accumulation does not inhibit xylose fermentation by engineered S. cerevisiae expressing xylA as severely as it inhibits the xylose isomerase reaction in vitro.     

Novel two‐stage fermentation process for bioethanol production using Saccharomyces pastorianus

Bioethanol produced from lignocellulosic materials has the potential to be economically feasible, if both glucose and xylose released from cellulose and hemicellulose can be efficiently converted to ethanol. Saccharomyces spp. can efficiently convert glucose to ethanol; however, xylose conversion to ethanol is a major hurdle due to lack of xylose‐metabolizing pathways. In this study, a novel two‐stage fermentation process was investigated to improve bioethanol productivity. In this process, xylose is converted into biomass via non‐Saccharomyces microorganism and coupled to a glucose‐utilizing Saccharomyces fermentation. Escherichia coli was determined to efficiently convert xylose to biomass, which was then killed to produce E. coli extract. Since earlier studies with Saccharomyces pastorianus demonstrated that xylose isomerase increased ethanol productivities on pure sugars, the addition of both E. coli extract and xylose isomerase to S. pastorianus fermentations on pure sugars and corn stover hydrolysates were investigated. It was determined that the xylose isomerase addition increased ethanol productivities on pure sugars but was not as effective alone on the corn stover hydrolysates. It was observed that the E. coli extract addition increased ethanol productivities on both corn stover hydrolysates and pure sugars. The ethanol productivities observed on the corn stover hydrolysates with the E. coli extract addition was the same as observed on pure sugars with both E. coli extract and xylose isomerase additions. These results indicate that the two‐stage fermentation process has the capability to be a competitive alternative to recombinant Saccharomyces cerevisiae‐based fermentations. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:300–310, 2014     

Lipid production from Yarrowia lipolytica Po1g grown in sugarcane bagasse hydrolysate

This study investigated the possibility of utilizing detoxified sugarcane bagasse hydrolysate (DSCBH) as an alternative carbon source to culture Yarrowia lipolytica Po1g for microbial oil and biodiesel production. Sugarcane bagasse hydrolysis with 2.5% HCl resulted in maximum total sugar concentration (21.38 g/L) in which 13.59 g/L is xylose, 3.98 g/L is glucose, and 2.78 g/L is arabinose. Detoxification of SCBH by Ca(OH)₂ neutralization reduced the concentration of 5-hydroxymethylfurfural and furfural by 21.31% and 24.84%, respectively. Growth of Y. lipolytica Po1g in DSCBH with peptone as the nitrogen source gave maximum biomass concentration (11.42 g/L) compared to NH₄NO₃ (6.49 g/L). With peptone as the nitrogen source, DSCBH resulted in better biomass concentration than d-glucose (10.19 g/L), d-xylose (9.89 g/L) and NDSCBH (5.88 g/L). The maximum lipid content, lipid yield and lipid productivity of Y. lipolytica Po1g grown in DSCBH and peptone was 58.5%, 6.68 g/L and 1.76 g/L-day, respectively.     

Biological pretreatment of corn stover with Phlebia brevispora NRRL‐13108 for enhanced enzymatic hydrolysis and efficient ethanol production

Biological pretreatment of lignocellulosic biomass by white‐rot fungus can represent a low‐cost and eco‐friendly alternative to harsh physical, chemical, or physico‐chemical pretreatment methods to facilitate enzymatic hydrolysis. In this work, solid‐state cultivation of corn stover with Phlebia brevispora NRRL‐13018 was optimized with respect to duration, moisture content and inoculum size. Changes in composition of pretreated corn stover and its susceptibility to enzymatic hydrolysis were analyzed. About 84% moisture and 42 days incubation at 28°C were found to be optimal for pretreatment with respect to enzymatic saccharification. Inoculum size had little effect compared to moisture level. Ergosterol data shows continued growth of the fungus studied up to 57 days. No furfural and hydroxymethyl furfural were produced. The total sugar yield was 442 ± 5 mg/g of pretreated corn stover. About 36 ± 0.6 g ethanol was produced from 150 g pretreated stover per L by fed‐batch simultaneous saccharification and fermentation (SSF) using mixed sugar utilizing ethanologenic recombinant Eschericia coli FBR5 strain. The ethanol yields were 32.0 ± 0.2 and 38.0 ± 0.2 g from 200 g pretreated corn stover per L by fed‐batch SSF using Saccharomyces cerevisiae D5A and xylose utilizing recombinant S. cerevisiae YRH400 strain, respectively. This research demonstrates that P. brevispora NRRL‐13018 has potential to be used for biological pretreatment of lignocellulosic biomass. This is the first report on the production of ethanol from P. brevispora pretreated corn stover. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:365–374, 2017     

Yeast strains for ethanol production from lignocellulosic hydrolysates during in situ detoxification

Yeast strains Y1, Y4 and Y7 demonstrated high conversion efficiencies for sugars and high abilities to tolerate or metabolize inhibitors in dilute-acid lignocellulosic hydrolysates. Strains Y1 and Y4 completely consumed the glucose within 24 h in dilute-acid lignocellulosic hydrolysate during in situ detoxification, and the maximum ethanol yields reached 0.49 g and 0.45 g ethanol/g glucose, equivalent to maximum theoretical values of 96% and 88.2%, respectively. Strain Y1 could metabolize xylose to xylitol with a yield of 0.64 g/g xylose, whereas Y4 was unable to utilize xylose as a substrate. Strain Y7 was able to consume sugars (glucose and xylose) within 72 h during hydrolysate in situ detoxification, producing a high ethanol yield (equivalent to 93.6% of the maximum theoretical value). Y1 and Y7 are the most efficient yeast strains yet reported for producing ethanol from non-detoxified dilute-acid lignocellulosic hydrolysates. These findings offer huge potential for improving the economics of bio-ethanol production from lignocellulosic hydrolysates.     

A C6/C5 co‐fermenting Saccharomyces cerevisiae strain with the alleviation of antagonism between xylose utilization and robustness

During second‐generation bioethanol production from lignocellulosic biomass, the desired traits for fermenting microorganisms, such as Saccharomyces cerevisiae, are high xylose utilization and high robustness to inhibitors in lignocellulosic hydrolysates. However, as observed previously, these two traits easily showed the antagonism, one rising and the other falling, in the C6/C5 co‐fermenting S. cerevisiae strain. In this study, LF1 obtained in our previous study is an engineered budding yeast strain with a superior co‐fermentation capacity of glucose and xylose, and was then mutated by atmospheric and room temperature plasma (ARTP) mutagenesis to improve its robustness. The ARTP‐treated cells were grown in 50% (v/v) leachate from lignocellulose pretreatment with high inhibitors content for adaptive evolution. After 30 days, the generated mutant LF1‐6 showed significantly enhanced tolerance, with a six‐fold increase in cell density in the above leachate. Unfortunately, its xylose utilization dropped markedly, indicating the recurrence of the negative correlation between xylose utilization and robustness. To alleviate this antagonism, LF1‐6 cells were iteratively mutated with ARTP mutagenesis and then anaerobically grown using xylose as the sole carbon source, and xylose utilization was restored in the resulting strain 6M‐15. 6M‐15 also exhibited increased co‐fermentation performance of xylose and glucose with the highest ethanol productivity reported to date (0.525 g g^?1 h^?1) in high‐level mixed sugars (80 g L^?1 glucose and 40 g L^?1 xylose) with no inhibitors. Meanwhile, its fermentation time was shortened by 8 h compared to that of LF1. During the fermentation of non‐detoxified lignocellulosic hydrolysate with high inhibitor concentrations at pH ~3.5, 6M‐15 can efficiently convert glucose and xylose with an ethanol yield of 0.43 g g^?1. 6M‐15 is also regarded as a potential chassis cell for further design of a customized strain suitable for production of second‐generation bioethanol or other high value‐added products from lignocellulosic biomass.     

Characterization of phenology,physiology, morphology and biomass traits across a broad Euro‐Mediterranean ecotypic panel of the lignocellulosic feedstock Arundo donax

Giant reed (Arundo donax L.) is a perennial rhizomatous grass, which has attracted great attention as a potential lignocellulosic feedstock for bioethanol production due to high biomass yield in marginal land areas, high polysaccharide content and low inhibitor levels in microbial fermentations. However, little is known about the trait variation that is available across a broad ecotypic panel of A. donax nor the traits that contribute most significantly to yield and growth in drought prone environments. A collection of 82 ecotypes of A. donax sampled across the Mediterranean basin was planted in a common garden experimental field in Savigliano, Italy. We analysed the collection using 367 clumps representing replicate plantings of 82 ecotypes for variation in 21 traits important for biomass accumulation and to identify the particular set of ecotypes with the most promising potential for biomass production. We measured morpho‐physiological, phenological and biomass traits and analysed causal relationships between traits and productivity characteristics assessed at leaf and canopy levels. The results identified differences among the 82 ecotypes for all studied traits: those showing the highest level of variability included stomatal resistance, stem density (StN), stem dry mass (StDM) and total biomass production (TotDM). Multiple regression analysis revealed that leaf area index, StDM, StN, number of nodes per stem, stem height and diameter were the most significant predictors of TotDM and the most important early selection criteria for bioenergy production from A. donax. These traits were used in a hierarchical cluster analysis to identify groups of similar ecotypes, and a selection was made of promising ecotypes for multiyear and multisite testing for biomass production. Heritability estimates were significant for all traits. The potential of this ecotype collection as a resource for studies of germplasm diversity and for the analysis of traits underpinning high productivity of A. donax is highlighted.     

Endophytic Fungal Strains of Soybean for Lipid Production

Lipids created via microbial biosynthesis are a potential raw material to replace plant-based oil for biodiesel production. Oleaginous microbial species currently available are capable of accumulating high amount of lipids in their cell biomass, but rarely can directly utilize lignocellulosic biomass as substrates. Thus this research focused on the screening and selection of new fungal strains that generate both lipids and hydrolytic enzymes. To search for oleaginous fungal strains in the soybean plant, endophytic fungi and fungi close to the plant roots were studied as a microbial source. Among 33 endophytic fungal isolates screened from the soybean plant, 13 have high lipid content (>20 % dry biomass weight); among 38 fungal isolates screened from the soil surrounding the soybean roots, 14 have high lipid content. Also, five fungal isolates with both high lipid content and promising biomass production were selected for further studies on their cell growth, oil accumulation, lipid content and profile, utilization of various carbon sources, and cellulase production. The results indicate that most strains could utilize different types of carbon sources and some strains accumulated >40 % of the lipids based on the dry cell biomass weight. Among these promising strains, some Fusarium strains specifically showed considerable production of cellulase, which offers great potential for biodiesel production by directly utilizing inexpensive lignocellulosic material as feedstock.