Production of high protein yeast using enzymatically liquefied almond hulls.

Animal feed ingredients, especially those abundant in high quality protein, are the most expensive component of livestock production. Sustainable alternative feedstocks may be sourced from abundant, low value agricultural byproducts. California almond production generates nearly 3 Mtons of biomass per year with about 50% in the form of hulls. Almond hulls are a low-value byproduct currently used primarily for animal feed for dairy cattle. However, the protein and essential amino acid content are low, at ~30% d.b.. The purpose of this study was to improve the protein content and quality using yeast. To achieve this, the almond hulls were liquefied to liberate soluble and structural sugars. A multi-phase screening approach was used to identify yeasts that can consume a large proportion of the sugars in almond hulls while accumulating high concentrations of amino acids essential for livestock feed. Compositional analysis showed that almond hulls are rich in polygalacturonic acid (pectin) and soluble sucrose. A pectinase-assisted process was optimized to liquefy and release soluble sugars from almond hulls. The resulting almond hull slurry containing solubilized sugars was subsequently used to grow high-protein yeasts that could consume nutrients in almond hulls while accumulating high concentrations of high-quality protein rich in essential amino acids needed for livestock feed, yielding a process that would produce 72 mg protein/g almond hull. Further work is needed to achieve conversion of galacturonic acid to yeast cell biomass.

Laboratory Screening Protocol to Identify Novel Oleaginous Yeasts.

Oleaginous microbes, which contain over 20% intracellular lipid, predominantly triacylglycerols (TG), by dry weight, have been discovered to have high oil content by many different protocols, ranging from simple staining to more complex chromatographic methods. In our laboratory, a methodical process was implemented to identify high oil yeasts, designed to minimize labor while optimizing success in identifying high oil strains among thousands of candidates. First, criteria were developed to select candidate yeast strains for analysis. These included observation of buoyancy of the yeast cell mass in 20% glycerol, and phylogenetic placement near known oleaginous species. A low-labor, semiquantitative Nile red staining protocol was implemented to screen numerous yeast cultures for high oil content in 96-well plates. Then, promising candidates were selected for more quantitative analysis. A more labor-intensive and quantitative gravimetric assay was implemented that gave consistent values for intracellular oil content for a broad range of yeast species. Finally, an LC-MS protocol was utilized to quantify and identify yeast triacylglycerols. This progressive approach was successful in identifying 30 new oleaginous yeast species, out of over 1000 species represented in the Phaff Yeast Culture Collection.

Extracellular fungal polyol lipids: A new class of potential high value lipids.

Extracellular fungal glycolipid biosurfactants have attracted attention because productivities can be high, cheap substrates can be used, the molecules are secreted into the medium and the downstream processing is relatively simple. Three classes of extracellular fungal glycolipid biosurfactants have provided most of the scientific advances in this area, namely sophorolipids, mannosylerythritol lipids and cellobioselipids. Polyol lipids, a fourth class of extracellular fungal glycolipid biosurfactants, comprise two groups of molecules: liamocins produced by the yeast-like fungus Aureobasidium pullulans, and polyol esters of fatty acids, produced by some Rhodotorula yeast species. Both are amphiphilic, surface active molecules with potential for commercial development as surfactants for industrial and household applications. The current knowledge of polyol lipids highlights an emerging group of extracellular fungal glycolipid biosurfactants and provides a perspective of what next steps are needed to harness the benefits and applications of this novel group of molecules.

1-Ethyl-3-methylimidazolium tolerance and intracellular lipid accumulation of 38 oleaginous yeast species.

Pretreatment with ionic liquids (IL) such as 1-ethyl-3-methylimidazolium chloride or acetate is an effective method for aiding deconstruction of lignocellulosic biomass; however, the residual IL remaining in hydrolysates can be inhibitory to growth of ethanologenic or oleaginous yeasts that have been examined in the literature. The aim of this study was to identify oleaginous yeasts that are tolerant of the IL [C2C1Im][OAc] and [C2C1Im]Cl using 45 strains belonging to 38 taxonomically diverse species within phyla Ascomycota and Basidiomycota. Yeasts were cultivated in laboratory medium supplemented with 0, 2, or 4% IL in 96-well plates. The eight most tolerant strains were then cultivated in 10-mL media with no IL, 242mM [C2C1Im][OAc], or 242mM [C2C1Im]Cl. The effects of [C2C1Im]+ exposure on cell mass production and lipid accumulation varied at the species and strain level. The acetate salt decreased cell biomass and lipid production more severely than did the chloride ion for six strains. Lipid output was not markedly different (2.1 vs. 2.3 g/L) in Yarrowia lipolytica UCDFST 51-30, but decreased from 5 to 65% in other yeasts. An equimolar concentration of the chloride salt resulted in much milder effects, from 25% decrease to 66% increase in lipid output. The highest lipid outputs in this media were 8.3 and 7.9 g/L produced by Vanrija humicola UCDFST 10-1004 and UCDFST 12-717, respectively. These results demonstrated substantial lipid production in the presence of [C2C1Im]Cl at concentrations found in lignocellulosic hydrolysates, and thus, these two strains are ideal candidates for further investigation.

Simultaneous production of intracellular triacylglycerols and extracellular polyol esters of fatty acids by Rhodotorula babjevae and Rhodotorula aff. paludigena.

Microbial oils have been analyzed as alternatives to petroleum. However, just a handful of microbes have been successfully adapted to produce chemicals that can compete with their petroleum counterparts. One of the reasons behind the low success rate is the overall economic inefficiency of valorizing a single product. This study presents a lab-scale analysis of two yeast species that simultaneously produce multiple high-value bioproducts: intracellular triacylglycerols (TG) and extracellular polyol esters of fatty acids (PEFA), two lipid classes with immediate applications in the biofuels and surfactant industries. At harvest, the yeast strain Rhodotorula aff. paludigena UCDFST 81-84 secreted 20.9 ± 0.2 g L-1 PEFA and produced 8.8 ± 1.0 g L-1 TG, while the yeast strain Rhodotorula babjevae UCDFST 04-877 secreted 11.2 ± 1.6 g L-1 PEFA and 18.5 ± 1.7 g L-1 TG. The overall glucose conversion was 0.24 and 0.22 g(total lipid) g (glucose)-1 , respectively. The results present a stable and scalable microbial growth platform yielding multiple co-products.

Discovery of synthesis and secretion of polyol esters of fatty acids by four basidiomycetous yeast species in the order Sporidiobolales.

Polyol esters of fatty acids (PEFA) are amphiphilic glycolipids produced by yeast that could play a role as natural, environmentally friendly biosurfactants. We recently reported discovery of a new PEFA-secreting yeast species, Rhodotorula babjevae, a basidiomycetous yeast to display this behavior, in addition to a few other Rhodotorula yeasts reported on the 1960s. Additional yeast species within the taxonomic order Sporidiobolales were screened for secreted glycolipid production. PEFA production equal or above 1 g L-1 were detected in 19 out of 65 strains of yeast screened, belonging to 6 out of 30 yeast species tested. Four of these species were not previously known to secrete glycolipids. These results significantly increase the number of yeast species known to secrete PEFA, holding promise for expanding knowledge of PEFA synthesis and secretion mechanisms, as well as setting the groundwork towards commercialization.

Multiplatform Mass Spectrometry-Based Approach Identifies Extracellular Glycolipids of the Yeast Rhodotorula babjevae UCDFST 04-877.

A multiplatform mass spectrometry-based approach was used for elucidating extracellular lipids with biosurfactant properties produced by the oleaginous yeast Rhodotorula babjevae UCDFST 04-877. This strain secreted 8.6 ± 0.1 g/L extracellular lipids when grown in a benchtop bioreactor fed with 100 g/L glucose in medium without addition of hydrophobic substrate, such as oleic acid. Untargeted reversed-phase liquid chromatography-quadrupole/time-of-flight mass spectrometry (QTOFMS) detected native glycolipid molecules with masses of 574-716 Da. After hydrolysis into the fatty acid and sugar components and hydrophilic interaction chromatography-QTOFMS analysis, the extracellular lipids were found to consist of hydroxy fatty acids and sugar alcohols. Derivatization and chiral separation gas chromatography-mass spectrometry (GC-MS) identified these components as d-arabitol, d-mannitol, (R)-3-hydroxymyristate, (R)-3-hydroxypalmitate, and (R)-3-hydroxystearate. In order to assemble these substructures back into intact glycolipids that were detected in the initial screen, potential structures were in-silico acetylated to match the observed molar masses and subsequently characterized by matching predicted and observed MS/MS fragmentation using the Mass Frontier software program. Eleven species of acetylated sugar alcohol esters of hydroxy fatty acids were characterized for this yeast strain.

Eighteen new oleaginous yeast species.

Of 1600 known species of yeasts, about 70 are known to be oleaginous, defined as being able to accumulate over 20 % intracellular lipids. These yeasts have value for fundamental and applied research. A survey of yeasts from the Phaff Yeast Culture Collection, University of California Davis was performed to identify additional oleaginous species within the Basidiomycota phylum. Fifty-nine strains belonging to 34 species were grown in lipid inducing media, and total cell mass, lipid yield and triacylglycerol profiles were determined. Thirty-two species accumulated at least 20 % lipid and 25 species accumulated over 40 % lipid by dry weight. Eighteen of these species were not previously reported to be oleaginous. Triacylglycerol profiles were suitable for biodiesel production. These results greatly expand the number of known oleaginous yeast species, and reveal the wealth of natural diversity of triacylglycerol profiles within wild-type oleaginous Basidiomycetes.

The United States Culture Collection Network (USCCN): Enhancing Microbial Genomics Research through Living Microbe Culture Collections.

The mission of the United States Culture Collection Network (USCCN; http://usccn.org) is "to facilitate the safe and responsible utilization of microbial resources for research, education, industry, medicine, and agriculture for the betterment of human kind." Microbial culture collections are a key component of life science research, biotechnology, and emerging global biobased economies. Representatives and users of several microbial culture collections from the United States and Europe gathered at the University of California, Davis, to discuss how collections of microorganisms can better serve users and stakeholders and to showcase existing resources available in public culture collections.

Yeast tolerance to the ionic liquid 1-ethyl-3-methylimidazolium acetate.

Lignocellulosic plant biomass is the target feedstock for production of second-generation biofuels. Ionic liquid (IL) pretreatment can enhance deconstruction of lignocellulosic biomass into sugars that can be fermented to ethanol. Although biomass is typically washed following IL pretreatment, small quantities of residual IL can inhibit fermentative microorganisms downstream, such as the widely used ethanologenic yeast, Saccharomyces cerevisiae. The aim of this study was to identify yeasts tolerant to the IL 1-ethyl-3-methylimidazolium acetate, one of the top performing ILs known for biomass pretreatment. One hundred and sixty eight strains spanning the Ascomycota and Basidiomycota phyla were selected for screening, with emphasis on yeasts within or closely related to the Saccharomyces genus and those tolerant to saline environments. Based on growth in media containing 1-ethyl-3-methylimidazolium acetate, tolerance to IL levels ranging 1-5% was observed for 80 strains. The effect of 1-ethyl-3-methylimidazolium acetate concentration on maximum cell density and growth rate was quantified to rank tolerance. The most tolerant yeasts included strains from the genera Clavispora, Debaryomyces, Galactomyces, Hyphopichia, Kazachstania, Meyerozyma, Naumovozyma, Wickerhamomyces, Yarrowia, and Zygoascus. These yeasts included species known to degrade plant cell wall polysaccharides and those capable of ethanol fermentation. These yeasts warrant further investigation for use in saccharification and fermentation of IL-pretreated lignocellulosic biomass to ethanol or other products.

Oleaginous yeasts for biodiesel: current and future trends in biology and production.

Production of biodiesel from edible plant oils is quickly expanding worldwide to fill a need for renewable, environmentally-friendly liquid transportation fuels. Due to concerns over use of edible commodities for fuels, production of biodiesel from non-edible oils including microbial oils is being developed. Microalgae biodiesel is approaching commercial viability, but has some inherent limitations such as requirements for sunlight. While yeast oils have been studied for decades, recent years have seen significant developments including discovery of new oleaginous yeast species and strains, greater understanding of the metabolic pathways that determine oleaginicity, optimization of cultivation processes for conversion of various types of waste plant biomass to oil using oleaginous yeasts, and development of strains with enhanced oil production. This review examines aspects of oleaginous yeasts not covered in depth in other recent reviews. Topics include the history of oleaginous yeast research, especially advances in the early 20th century; the phylogenetic diversity of oleaginous species, beyond the few species commonly studied; and physiological characteristics that should be considered when choosing yeast species and strains to be utilized for conversion of a given type of plant biomass to oleochemicals. Standardized terms are proposed for units that describe yeast cell mass and lipid production.

Identification of oleaginous yeast strains able to accumulate high intracellular lipids when cultivated in alkaline pretreated corn stover.

Microbial oil is a potential alternative to food/plant-derived biodiesel fuel. Our previous screening studies identified a wide range of oleaginous yeast species, using a defined laboratory medium known to stimulate lipid accumulation. In this study, the ability of these yeasts to grow and accumulate lipids was further investigated in synthetic hydrolysate (SynH) and authentic ammonia fiber expansion (AFEX™)-pretreated corn stover hydrolysate (ACSH). Most yeast strains tested were able to accumulate lipids in SynH, but only a few were able to grow and accumulate lipids in ACSH medium. Cryptococcus humicola UCDFST 10-1004 was able to accumulate as high as 15.5 g/L lipids, out of a total of 36 g/L cellular biomass when grown in ACSH, with a cellular lipid content of 40 % of cell dry weight. This lipid production is among the highest reported values for oleaginous yeasts grown in authentic hydrolysate. Preculturing in SynH media with xylose as sole carbon source enabled yeasts to assimilate both glucose and xylose more efficiently in the subsequent hydrolysate medium. This study demonstrates that ACSH is a suitable medium for certain oleaginous yeasts to convert lignocellullosic sugars to triacylglycerols for production of biodiesel and other valuable oleochemicals.

Accelerated degradation of exogenous indole by Burkholderia unamae strain CK43B exposed to pyrogallol-type polyphenols.

In modified Winogradsky's (MW) medium supplemented with excessive indole (1), Burkholderia unamae strain CK43B isolated from polyphenol-rich Shorea rhizosphere showed almost no cell growth, but it showed drastic cell growth given further supplementation of gallic acid, a simple plant polyphenol. This active cell growth of B. unamae CK43B was due to the stimulating effect of gallic acid on 1-degradation of bacterial cells, which acquired a nitrogen source in 1. Under aerobic culture conditions with appropriate concentrations (0.5-2.0 mM) of gallic acid, B. unamae CK43B started to decompose exogenous 1 in a dose-dependent manner, and finally accumulated catechol (5) via anthranilic acid (4). Pyrogallol also showed a cometabolic effect on decarboxylation-coupled oxidative deamination of B. unamae CK43B, producing 5 from 4, as gallic acid did. These results suggest that pyrogallol-type plant polyphenols act as stimulators on B. unamae CK43B, causing it to degrade an N-heterocyclic aromatic compound (NHAC) including nitrogen-containing humic substances.

Manipulation of culture conditions alters lipid content and fatty acid profiles of a wide variety of known and new oleaginous yeast species.

Oleaginous yeasts have been studied for oleochemical production for over 80 years. Only a few species have been studied intensely. To expand the diversity of oleaginous yeasts available for lipid research, we surveyed a broad diversity of yeasts with indicators of oleaginicity including known oleaginous clades, and buoyancy. Sixty-nine strains representing 17 genera and 50 species were screened for lipid production. Yeasts belonged to Ascomycota families, Basidiomycota orders, and the yeast-like algal genus Prototheca. Total intracellular lipids and fatty acid composition were determined under different incubation times and nitrogen availability. Thirteen new oleaginous yeast species were discovered, representing multiple ascomycete and basidiomycete clades. Nitrogen starvation generally increased intracellular lipid content. The fatty acid profiles varied with the growth conditions regardless of taxonomic affiliation. The dominant fatty acids were oleic acid, palmitic acid, linoleic acid, and stearic acid. Yeasts and culture conditions that produced fatty acids appropriate for biodiesel were identified.

Distinct metabolites for photoreactive L-phenylalanine derivatives in Klebsiella sp. CK6 isolated from rhizosphere of a wild dipterocarp sapling.

Photoaffinity labeling is a reliable analytical method for biological functional analysis. Three major photophores--aryl azide, benzophenone and trifluoromethyldiazirine--are utilized in analysis. Photophore-bearing L-phenylalanine derivatives, which are used for biological functional analysis, were inoculated into a Klebsiella sp. isolated from the rhizosphere of a wild dipterocarp sapling in Central Kalimantan, Indonesia, under nitrogen-limiting conditions. The proportions of metabolites were quite distinct for each photophore. These results indicated that photophores affected substrate recognition in rhizobacterial metabolic pathways, and differential photoaffinity labeling could be achieved using different photophore-containing L-phenylalanine derivatives.

Induction of biofilm formation in the betaproteobacterium Burkholderia unamae CK43B exposed to exogenous indole and gallic acid.

Burkholderia unamae CK43B, a member of the Betaproteobacteria that was isolated from the rhizosphere of a Shorea balangeran sapling in a tropical peat swamp forest, produces neither indole nor extracellular polymeric substances associated with biofilm formation. When cultured in a modified Winogradsky's medium supplemented with up to 1.7 mM indole, B. unamae CK43B maintains its planktonic state by cell swelling and effectively degrades exogenous indole. However, in medium supplemented with 1.7 mM exogenous indole and 1.0 mM gallic acid, B. unamae CK43B produced extracellular polymeric substances and formed a biofilm. The concentration indicated above of gallic acid alone had no effect on either the growth or the differentiation of B. unamae CK43B cells above a certain concentration threshold, whereas it inhibited indole degradation by B. unamae CK43B to 3-hydroxyindoxyl. In addition, coculture of B. unamae CK43B with indole-producing Escherichia coli in nutrient-rich Luria-Bertani medium supplemented with 1.0 mM gallic acid led to the formation of mixed cell aggregates. The viability and active growth of B. unamae CK43B cells in a coculture system with Escherichia coli were evidenced by fluorescence in situ hybridization. Our data thus suggest that indole facilitates intergenus communication between indole-producing gammaproteobacteria and some indole-degrading bacteria, particularly in gallic acid-rich environments.

Salkowski's reagent test as a primary screening index for functionalities of rhizobacteria isolated from wild dipterocarp saplings growing naturally on medium-strongly acidic tropical peat soil.

Rhizobacteria isolated from wild dipterocarp saplings in Central Kalimantan, Indonesia, were subjected to Salkowski's reagent test, which is often used in detecting indolic substances. Among 69 isolates grown in a low-nitrogen medium supplemented with L-tryptophan (TRP), culture fluids of 29 strains were positive to the test, in which 17 bacteria turned red and other 10 pink. All the red type rhizobacteria actively converted TRP into tryptophol (TOL), while some yielded indole-3-acetic acid (IAA) with TOL production. They also showed a capacity to decompose gallotannin into pyrogallol via gallic acid. On the other hand, an active IAA-producing Serratia sp. CK67, and three Fe-solubilizing Burkholderia spp. CK28, CK43, and Citrobacter sp. CK42, were all involved in pink type rhizobacteria, which were more effective, oxidative TRP-degraders than the red type rhizobacteria. Thus, Salkowski's reagent test should be a useful primary index in the screening of functional rhizobacteria in peatland ecosystem.