Low Initial Cell Density Research Articles

Erg251 has complex and pleiotropic effects on sterol composition, azole susceptibility, filamentation, and stress response phenotypes.

Ergosterol is essential for fungal cell membrane integrity and growth, and numerous antifungal drugs target ergosterol. Inactivation or modification of ergosterol biosynthetic genes can lead to changes in antifungal drug susceptibility, filamentation and stress response. Here, we found that the ergosterol biosynthesis gene ERG251 is a hotspot for point mutations during adaptation to antifungal drug stress within two distinct genetic backgrounds of Candida albicans. Heterozygous point mutations led to single allele dysfunction of ERG251 and resulted in azole tolerance in both genetic backgrounds. This is the first known example of point mutations causing azole tolerance in C. albicans. Importantly, single allele dysfunction of ERG251 in combination with recurrent chromosome aneuploidies resulted in bona fide azole resistance. Homozygous deletions of ERG251 caused increased fitness in low concentrations of fluconazole and decreased fitness in rich medium, especially at low initial cell density. Homozygous deletions of ERG251 resulted in accumulation of ergosterol intermediates consistent with the fitness defect in rich medium. Dysfunction of ERG251, together with FLC exposure, resulted in decreased accumulation of the toxic sterol (14-ɑ-methylergosta-8,24(28)-dien-3β,6α-diol) and increased accumulation of non-toxic alternative sterols. The altered sterol composition of the ERG251 mutants had pleiotropic effects on transcription, filamentation, and stress responses including cell membrane, osmotic and oxidative stress. Interestingly, while dysfunction of ERG251 resulted in azole tolerance, it also led to transcriptional upregulation of ZRT2, a membrane-bound Zinc transporter, in the presence of FLC, and overexpression of ZRT2 is sufficient to increase azole tolerance in wild-type C. albicans. Finally, in a murine model of systemic infection, homozygous deletion of ERG251 resulted in decreased virulence while the heterozygous deletion mutants maintain their pathogenicity. Overall, this study demonstrates that single allele dysfunction of ERG251 is a recurrent and effective mechanism of acquired azole tolerance. We propose that altered sterol composition resulting from ERG251 dysfunction mediates azole tolerance as well as pleiotropic effects on stress response, filamentation and virulence.

Low Initial Cell Density Promotes the Differentiation and Maturation of Human Pluripotent Stem Cells into Erythrocytes.

Human pluripotent stem cell (hPSC)-derived red blood cells (RBCs) possess great potential for compensating shortages in transfusion medicine. For better RBC generation from hPSCs, we compared the cell seeding density in the embryoid body formation-based hPSC induction protocol. In the selection of low- and high-density inoculation conditions, we found that low-density culture performed better in the final RBC product with more cell output and increased average cellular hemoglobin content. An elaborate study using flow cytometry demonstrated that low inoculation density promoted endothelial-to-hematopoietic transition, followed by improved hematopoietic progenitor formation and erythrocyte generation. The improved transformation from glycolysis to mitochondrial oxidation and reduced apoptosis might be responsible for this effect. Hints from RNA sequencing suggested that molecules involved in microenvironment interaction and metabolic regulation might respond for the different developmental potential. The possible mediators between outer message and intracellular response could be the nutrition sensors FOXO, PRKAA1 (AMPK), and MTOR genes. It is possible that low inoculation density triggered metabolic regulation signals, promoted mitochondrial oxidation, and resulted in enhanced cell amplification and hematopoietic differentiation. The low cell culture density will improve RBC generation from hPSCs.

Exploring kinetics, removal mechanism and possible transformation products of tigecycline by Chlorella pyrenoidosa

The accumulation of antibiotics in wastewater leads to broad antibiotic resistance, threating human health. Microalgae have been receiving attention due to their ability to remove antibiotics from wastewater. Tigecycline (TGC) is a broad-spectrum glycylcycline antibiotic. It has not been investigated for removal by microalgae. The removal kinetics of TGC by Chlorella pyrenoidosa were evaluated under different initial dry cell densities, TGC concentrations, temperatures and light intensity conditions. Approximately 90% of TGC could be removed when the TGC concentration was 10 mg∙L−1 and the initial dry cell density was more than 0.2 g∙L−1. A low value of TGC per g dry cell weight ratio led to a high removal efficiency of TGC. The initial dry cell density of microalgae was also critical for the removal of TGC. A high initial dry cell density is better than a low initial dry cell density to remove TGC when the ratio of the TGC concentration to dry cell weight are the same at the beginning of the cultivation. The removal mechanisms were investigated. Photolysis was a slow process that did not lead to removal at the beginning. Adsorption, hydrolysis, photolysis and biodegradation by microalgae were the main contributors to the removal of TGC. TGC was easily hydrolyzed under high -temperature conditions. Three transformation products of TGC by microalgae were identified. The stability of TGC was evaluated in water and salt solutions of citric acid, K2HPO4·3H2O and ferric ammonium citrate. TGC was stable in ultrapure water and citric acid solution. TGC was hydrolyzed in K2HPO4·3H2O and ferric ammonium citrate solutions.

Pathogenicity and antibiotic susceptibility of two bacterial pathogens associated with the clam Tapes decussatus in some Egyptian fisheries

Bivalve aquaculture is generally influenced by bacterial pathogens that cause high mortality-related losses in hatcheries. Six bacterial phenotypes had been previously isolated as the most predominant microorganisms in carpet shell clam ( Tapes decussates ) samples collected from natural beds in representative Egyptian clam fisheries. The main aims of this study were to investigate the nature of the pathogenic strains that significantly affect clam survival and test their susceptibility to commercially available antibiotics. Based on their 16S rRNA sequences and some biochemical features, two potent clam pathogens were emerged; one of which is gram-positive and the other is gram-negative. The former isolate was identified as Micrococcus luteus and the latter as Vibrio alginolyticus . Experimental challenges with the two bacterial pathogens introduced at different initial cell concentrations (2.5 X 10 4 - 7.5 X 10 4 cfu ml -1 ) showed markedly diverse clam mortality results. However, the pathogenic interaction of M. luteus with clam survival was generally higher than that of V. alginolyticus . When introduced at a relatively low initial cell density, the infectious records of V. alginolyticus were significantly increased suggesting that the expression of its key virulence factors is mainly triggered as a response to host contact. Antibiotic susceptibility tests suggested chloramphenicol and tetracycline as markedly effective agents that can be used to control the spread of these two bacterial pathogens in aquaculture applications.

Characterization and Optimization of a Fermentation Process for the Production of High Cell Densities and Lipids Using Heterotrophic Cultivation of Chlorella protothecoides

Abstract This article reports an in-depth characterization and optimization of a fermentation process with the aim of increasing the production of high cell densities as a prerequisite for high productivity of lipids. The freshwater microalgae Chlorella protothecoides was grown heterotrophically in a bioreactor on aerated and stirred cultivation medium with glucose as a carbon source and yeast extract as a nitrogen source. The detailed effect of carbon to nitrogen (C/N) ratio on growth and lipid content of C. protothecoides was studied. Results show that with a C/N ratio lower than 12, the medium was carbon-limited, favoring cell proliferation, and under nitrogen-starvation conditions, (higher C/N ratios), lipid accumulation was enhanced. A two-stage, fed-batch process was developed and achieved a maximum of 255.3 g/L of biomass over 228 h of fermentation, from a low initial cell density (< 2 g/L). The specific growth rate, measured when C/N ratio was low, was 0.078 h−1. The lipid productivity was 16.7 g/...

Physiological responses to acid stress by Saccharomyces cerevisiae when applying high initial cell density.

High initial cell density is used to increase volumetric productivity and shorten production time in lignocellulosic hydrolysate fermentation. Comparison of physiological parameters in high initial cell density cultivation of Saccharomyces cerevisiae in the presence of acetic, formic, levulinic and cinnamic acids demonstrated general and acid-specific responses of cells. All the acids studied impaired growth and inhibited glycolytic flux, and caused oxidative stress and accumulation of trehalose. However, trehalose may play a role other than protecting yeast cells from acid-induced oxidative stress. Unlike the other acids, cinnamic acid did not cause depletion of cellular ATP, but abolished the growth of yeast on ethanol. Compared with low initial cell density, increasing initial cell density reduced the lag phase and improved the bioconversion yield of cinnamic acid during acid adaptation. In addition, yeast cells were able to grow at elevated concentrations of acid, probable due to the increase in phenotypic cell-to-cell heterogeneity in large inoculum size. Furthermore, the specific growth rate and the specific rates of glucose consumption and metabolite production were significantly lower than at low initial cell density, which was a result of the accumulation of a large fraction of cells that persisted in a viable but non-proliferating state.

High concentration CO2 sequestration by using microalgae in staged cultivation

The acclimated fresh water microalga Chlorella vulgaris has the excellent growing capability in the process of staged cultivation (SC). In this study, the alga solutions with low (106/mL) and high (107/m) cell densities showed different abilities of growth with different levels of CO2–air mixture gases[5%, 10%, 15% (v/v)]. The SC process can improve the algal mass productivity rapidly by the aerating increased concentrations of CO2 of mixed gases into the algae solution in a short time. The results showed that the cell density was improved to 1.72 g L−1 within 7 days, showing 3.1, 1.48, and 1.70 times improvement compared to that in the biomass from the culture with separate 5, 10, and 15% CO2–air mixture gases. Besides, there were not the obvious distinctions on the contents of biochemicals (carbohydrate, protein, and lipid) from these algae. This study proved that SC process was an efficient method to improve biomass productivity of Chlorella sp. in a short time with low initial cell density. © 2016 American Institute of Chemical Engineers Environ Prog, 35: 1862–1867, 2016

Isolation, identification, and algicidal activity of aerobic denitrifying bacterium R11 and its effect on Microcystis aeruginosa.

Recently, algicidal bacteria have attracted attention as possible agents for the inhibition of algal water blooms. In this study, an aerobic denitrifying bacterium, R11, with high algicidal activity against the toxic Microcystis aeruginosa was isolated from lake sediments. Based on its physiological characteristics and 16S rRNA gene sequence, it was identified as Raoultella, indicating that the bacterium R11 has a good denitrifying ability at 30 °C and can reduce the concentration of nitrate-N completely within 36 h. Additionally, different algicidal characteristics against Microcystis aeruginosa were tested. The results showed that the initial bacterial cell density and algal cell densities strongly influence the removal rates of chlorophyll a. Algicidal activity increased with an increase in the bacterial cell density. With densities of bacterial culture at over 2.4 × 10(5) cell/mL, algicidal activity of up to 80% was obtained in 4 days. We have demonstrated that, with the low initial algal cell density (OD680 less than 0.220), the algicidal activity reached was higher than 90% after 6 days.

The Effects of Initial Cell Density on the Growth and Proliferation of the Potentially Toxic Cyanobacterium &amp;lt;i&amp;gt;Microcystis aeruginosa&amp;lt;/i&amp;gt;

Increasing input of nutrients is leading to significant eutrophication in aquatic systems across the globe. Toxin production in those blooms is correlated with high cell density. A study was done to investigate the effects of initial cell density on the growth rate of the potentially toxic cyanobacterium Microcystis aeruginosa, in unfiltered lake water as well a common algal growth medium under laboratory conditions. Five treatments were established in the laboratory varying the initial cell density of M. aeruginosa and the growth medium. Unfiltered lake water was inoculated with a low, medium, and high initial cell density and Bold’s medium was inoculated with low and high initial cell densities. There was a significant difference in final cell density between the lake water treatments with the high inoculum of cell treatment reaching the highest final cell density. There was also a significant difference in final cell density between the Bold’s medium treatments. These results indicate there is a relationship between initial cell density of M. aeruginosa and final cell density, however, the results are similar when nutrients are available. Diverse algal communities can keep toxin producing algae in low density therefore precluding the need of toxin production.

Evolved strains of Scheffersomyces stipitis achieving high ethanol productivity on acid- and base-pretreated biomass hydrolyzate at high solids loading.

BackgroundLignocellulosic biomass is an abundant, renewable feedstock useful for the production of fuel-grade ethanol via the processing steps of pretreatment, enzyme hydrolysis, and microbial fermentation. Traditional industrial yeasts do not ferment xylose and are not able to grow, survive, or ferment in concentrated hydrolyzates that contain enough sugar to support economical ethanol recovery since they are laden with toxic byproducts generated during pretreatment.ResultsRepetitive culturing in two types of concentrated hydrolyzates was applied along with ethanol-challenged xylose-fed continuous culture to force targeted evolution of the native pentose fermenting yeast Scheffersomyces (Pichia) stipitis strain NRRL Y-7124 maintained in the ARS Culture Collection, Peoria, IL. Isolates collected from various enriched populations were screened and ranked based on relative xylose uptake rate and ethanol yield. Ranking on hydrolyzates with and without nutritional supplementation was used to identify those isolates with best performance across diverse conditions.ConclusionsRobust S. stipitis strains adapted to perform very well in enzyme hydrolyzates of high solids loading ammonia fiber expansion-pretreated corn stover (18% weight per volume solids) and dilute sulfuric acid-pretreated switchgrass (20% w/v solids) were obtained. Improved features include reduced initial lag phase preceding growth, significantly enhanced fermentation rates, improved ethanol tolerance and yield, reduced diauxic lag during glucose-xylose transition, and ability to accumulate >40 g/L ethanol in <167 h when fermenting hydrolyzate at low initial cell density of 0.5 absorbance units and pH 5 to 6.

Morphology of &amp;lt;i&amp;gt;Emiliania huxleyi&amp;lt;/i&amp;gt; coccoliths on the northwestern European shelf – is there an influence of carbonate chemistry?

Abstract. Within the context of the UK Ocean Acidification project, Emiliania huxleyi (type A) coccolith morphology was examined from samples collected during cruise D366. In particular, a morphometric study of coccolith size and degree of calcification was made on scanning electron microscope images of samples from shipboard CO2 perturbation experiments and from a set of environmental samples with significant variation in calcite saturation state (Ωcalcite). One bioassay in particular (E4 from the southern North Sea) yielded unambiguous results – in this bioassay exponential growth from a low initial cell density occurred with no nutrient enrichment and coccosphere numbers increased tenfold during the experiment. The samples with elevated CO2 saw significantly reduced coccolithophore growth. However, coccolithophore morphology was not significantly affected by the changing CO2 conditions even under the highest levels of perturbation (1000 μatm CO2). Environmental samples similarly showed no correlation of coccolithophore morphology with calcite saturation state. Some variation in coccolith size and degree of calcification does occur but this seems to be predominantly due to genotypic differentiation between populations on the shelf and in the open ocean.

Density of founder cells affects spatial pattern formation and cooperation in Bacillus subtilis biofilms.

In nature, most bacteria live in surface-attached sedentary communities known as biofilms. Biofilms are often studied with respect to bacterial interactions. Many cells inhabiting biofilms are assumed to express 'cooperative traits', like the secretion of extracellular polysaccharides (EPS). These traits can enhance biofilm-related properties, such as stress resilience or colony expansion, while being costly to the cells that express them. In well-mixed populations cooperation is difficult to achieve, because non-cooperative individuals can reap the benefits of cooperation without having to pay the costs. The physical process of biofilm growth can, however, result in the spatial segregation of cooperative from non-cooperative individuals. This segregation can prevent non-cooperative cells from exploiting cooperative neighbors. Here we examine the interaction between spatial pattern formation and cooperation in Bacillus subtilis biofilms. We show, experimentally and by mathematical modeling, that the density of cells at the onset of biofilm growth affects pattern formation during biofilm growth. At low initial cell densities, co-cultured strains strongly segregate in space, whereas spatial segregation does not occur at high initial cell densities. As a consequence, EPS-producing cells have a competitive advantage over non-cooperative mutants when biofilms are initiated at a low density of founder cells, whereas EPS-deficient cells have an advantage at high cell densities. These results underline the importance of spatial pattern formation for competition among bacterial strains and the evolution of microbial cooperation.

Sustained hydrogen photoproduction by phosphorus-deprived Chlamydomonas reinhardtii cultures

This study demonstrates that, besides sulfur deprivation, sustained H2 photoproduction in Chlamydomonas reinhardtii cultures can be generated by incubating algae under phosphorus-deprived (−P) conditions. However, phosphorus deficiency in algal cells could not be obtained by resuspension of algae in −P medium, evidently due to a significant reserve of phosphorus in cells. In this study, phosphorus deficiency was accomplished by inoculating the washed algae into the −P medium at low initial cell densities (below 2 mg Chl l−1). After the initial growth period, where cells utilize intracellular phosphorus, algae established anaerobic environment followed by the period of H2 photoproduction. The maximum H2 output (∼70 ml l−1) was obtained in cultures with the initial Chl content ∼1 mg l−1. Cultures with Chl above 2 mg l−1 did not produce H2 gas. The physiological response of algal cultures to phosphorus deprivation demonstrated significant similarities with the response of algae to sulfur depletion.

In situ monitoring of IncF plasmid transfer on semi-solid agar surfaces reveals a limited invasion of plasmids in recipient colonies

Most natural conjugative IncF plasmids encode a fertility inhibition system that represses transfer gene expression in the majority of plasmid-carrying cells. The successful spread of these plasmids in clinically relevant bacteria has been suggested to be supported by a transitory derepression of transfer gene expression in newly formed transconjugants. In this study, we aimed to monitor the extent of transitory derepression during agar surface matings in situ by comparing plasmid spread of the IncF plasmid R1 and its derepressed mutant R1drd19 at low initial cell densities. A zygotic induction strategy was used to visualize the spatial distribution of fluorescent transconjugants within the heterogeneous environment. Epifluorescence and confocal microscopy revealed different transfer patterns for both plasmids, however, spread beyond the first five recipient cell layers adjacent to the donor cells was not observed. Similar results were observed for other prototypical conjugative plasmids. These results cannot rule out that transitory derepression contributes to the limited R1 plasmid invasion, but other factors like nutrient availability or spatial structure seem to limit plasmid spread.

Designing optimal calcium phosphate scaffold–cell combinations using an integrative model-based approach

Bone formation is a very complex physiological process, involving the participation of many different cell types and regulated by countless biochemical, physical and mechanical factors, including naturally occurring or synthetic biomaterials. For the latter, calcium phosphate (CaP)-based scaffolds have proven to stimulate bone formation, but at present still result in a wide range of in vivo outcomes, which is partly related to the suboptimal use and combination with osteogenic cells. To optimize CaP scaffold selection and make their use in combination with cells more clinically relevant, this study uses an integrative approach in which mathematical modeling is combined with experimental research. This paper describes the development and implementation of an experimentally informed bioregulatory model of the effect of calcium ions released from CaP-based biomaterials on the activity of osteogenic cells and mesenchymal stem cell driven ectopic bone formation. The amount of bone formation predicted by the mathematical model corresponds to the amount measured experimentally under similar conditions. Moreover, the model is also able to qualitatively predict the experimentally observed impaired bone formation under conditions such as insufficient cell seeding and scaffold decalcification. A strategy was designed in silico to overcome the negative influence of a low initial cell density on the bone formation process. Finally, the model was applied to design optimal combinations of calcium-based biomaterials and cell culture conditions with the aim of maximizing the amount of bone formation. This work illustrates the potential of mathematical models as research tools to design more efficient and cell-customized CaP scaffolds for bone tissue engineering applications.

Growth of the Cyanobacterium Synechococcus leopoliensis CCAP1405/1 on Agar Media in the Presence of Heterotrophic Bacteria

The cyanobacterium Synechococcus leopoliensis CCAP1405/1 does not grow on common solid media made of agar, agarose HT, noble agar, gelrite and gelatin, although it grows in liquid media with the same components. The inoculation of S. leopoliensis CCAP1405/1 at a high initial cell density allowed it to grow on the agar media, and co-inoculation with one of the heterotrophic bacterial strains belonging to a wide range of phylogeny, showed the same effects even at a low initial cell density of S. leopoliensis CCAP1405/1. The addition of thiosulfate and high concentrations of vitamin B(12), biotin and thiamine also supported growth on solid media, but catalase had no effect. On inorganic solid media, the autotrophic cyanobacterial strain supported the growth of heterotrophic bacteria, suggesting mutual interaction.

Mammalian cell growth versus biofilm formation on biomaterial surfaces in an in vitro post-operative contamination model

Biomaterial-associated infections are the major cause of implant failure and can develop many years after implantation. Success or failure of an implant depends on the balance between host tissue integration and bacterial colonization. Here, we describe a new in vitro model for the post-operative bacterial contamination of implant surfaces and investigate the effects of contamination on the balance between mammalian cell growth and bacterial biofilm formation. U2OS osteosarcoma cells were seeded on poly(methyl methacrylate) in different densities and allowed to grow for 24&emsp14;h in a parallel-plate flow chamber at a low shear rate (0.14&emsp14;s(-1)), followed by contamination with Staphylococcus epidermidis ATCC 35983 at a shear rate of 11&emsp14;s(-1). The U2OS cells and staphylococci were allowed to grow simultaneously for another 24&emsp14;h under low-shear conditions (0.14&emsp14;s(-1)). Mammalian cell growth was severely impaired when the bacteria were introduced to surfaces with a low initial cell density (2.5 × 10(4) cells cm(-2)), but in the presence of higher initial cell densities (8.2 × 10(4) cells cm(-2) and 17 × 10(4) cells cm(-2)), contaminating staphylococci did not affect cell growth. This study is believed to be the first to show that a critical coverage by mammalian cells is needed to effectively protect a biomaterial implant against contaminating bacteria.

Optimization of enzymatic hydrolysis and ethanol fermentation from AFEX-treated rice straw

An abundant agricultural residue, rice straw (RS) was pretreated using ammonia fiber expansion (AFEX) process with less than 3% sugar loss. Along with commercial cellulase (Spezyme CP) at 15 filter paper unit/g of glucan, the addition of Multifect Xylanase at 2.67 mg protein/g glucan and Multifect Pectinase at 3.65 mg protein/g glucan was optimized to greatly increase sugar conversion of AFEX-treated RS. During enzymatic hydrolysis even at 6% glucan loading (equivalent to 17.8% solid loading), about 80.6% of glucan and 89.6% of xylan conversions (including monomeric and oligomeric sugars) were achieved. However, oligomeric glucose and xylose accounted for 12.3% of the total glucose and 37.0% of the total xylose, respectively. Comparison among the three ethanologenic strains revealed Saccharomyces cerevisiae 424A(LNH-ST) to be a promising candidate for RS hydrolysate with maximum ethanol metabolic yield of 95.3% and ethanol volumetric productivity of 0.26 g/L/h. The final concentration of ethanol at 37.0 g/L was obtained by S. cerevisiae 424A(LNH-ST) even with low cell density inoculum. A biorefinery combining AFEX pretreatment with S. cerevisiae 424A(LNH-ST) in separate hydrolysis and fermentation could achieve 175.6 g EtOH/kg untreated rice straw at low initial cell density (0.28 g dw/L) without washing pretreated biomass, detoxification, or nutrient supplementation.

Aeromonas–Acanthamoeba interaction and early shift to a viable but nonculturable state of Aeromonas by Acanthamoeba

To investigate the hypothesis that amoeba may comprise a significant environmental reservoir for Aeromonas, Acanthamoeba-Aeromonas interaction experiments were performed. Acanthamoeba were grown in monoculture and co-cultures with three different species of Aeromonas. Survival, invasion and viable but nonculturable state experiments were performed. We showed that at a low initial bacterial cell density, growth of Aeromonas spp. was inhibited by Acanthamoeba castellanii, while A. castellanii growth was unaffected. In contrast, a high initial bacterial cell density, Aeromonas hydrophila AEW44 and Aeromonas veronii biovar sobria AEW104 suppressed the growth of A. castellanii. Fluorescent and phase-contrast microscopic observations of GFP tagged Aer. hydrophila AEW44 demonstrated that the bacterial cells aggregated on A. castellanii cells after 15 min of incubation and internalized. Aeromonas hydrophila AEW44 cells were found to be actively moving. Interestingly, Aer. hydrophila AEW44 cells shifted more rapidly to a viable but nonculturable form when co-cultured with A. castellanii than in monoculture. We demonstrated that Aeromonas spp. are able to interact with and to infect the protozoan A. castellanii under laboratory conditions. Free-living amoeba might play a role as reservoir for Aeromonas, and thus may increase the transmission of Aeromonas by acting as a vehicle.

Ethanolic fermentation of hydrolysates from ammonia fiber expansion (AFEX) treated corn stover and distillers grain without detoxification and external nutrient supplementation

External nutrient supplementation and detoxification of hydrolysate significantly increase the production cost of cellulosic ethanol. In this study, we investigated the feasibility of fermenting cellulosic hydrolysates without washing, detoxification or external nutrient supplementation using ethanologens Escherichia coli KO11 and the adapted strain ML01 at low initial cell density (16 mg dry weight/L). The cellulosic hydrolysates were derived from enzymatically digested ammonia fiber expansion (AFEX)-treated corn stover and dry distiller's grain and solubles (DDGS) at high solids loading (18% by weight). The adaptation was achieved through selective evolution of KO11 on hydrolysate from AFEX-treated corn stover. All cellulosic hydrolysates tested (36-52 g/L glucose) were fermentable. Regardless of strains, metabolic ethanol yields were near the theoretical limit (0.51 g ethanol/g consumed sugar). Volumetric ethanol productivity of 1.2 g/h/L was achieved in fermentation on DDGS hydrolysate and DDGS improved the fermentability of hydrolysate from corn stover. However, enzymatic hydrolysis and xylose utilization during fermentation were the bottlenecks for ethanol production from corn stover at these experimental conditions. In conclusion, fermentation under the baseline conditions was feasible. Utilization of nutrient-rich feedstocks such as DDGS in fermentation can replace expensive media supplementation.