Inhibitor tolerance and flocculation of a yeast strain suitable for second generation bioethanol production

Background: Robust second generation bioethanol processes require microorganisms able to ferment inhibitory lignocellullosic hydrolysates. In this study, the inhibitor tolerance and flocculation characteristics of Saccharomyces cerevisiae CCUG53310 were evaluated in comparison with S. cerevisiae CBS8066. Results: The flocculating strain CCUG53310 could rapidly ferment all hexoses in dilute acid spruce hydrolysate, while CBS8066 was strongly inhibited in this medium. In synthetic inhibitory media, CCUG53310 was more tolerant to carboxylic acids and furan aldehydes, but more sensitive than CBS8066 to phenolic compounds. Despite the higher tolerance, the increase in expression of the YAP1, ATR1 and FLR1 genes, known to confer resistance to lignocellulose-derived inhibitors, was generally smaller in CCUG53310 than in CBS8066 in inhibitory media. The flocculation of CCUG53310 was linked to the expression of FLO8, FLO10 and one or more of FLO1, FLO5 or FLO9. Flocculation depended on cell wall proteins and Ca2+ ions, but was almost unaffected by other compounds and pH values typical for lignocellulosic media. Conclusions: S. cerevisiae CCUG53310 can be characterised as being very robust, with great potential for industrial fermentation of lignocellulosic hydrolysates relatively low in phenolic inhibitors

Inhibitor tolerance and flocculation of a yeast strain suitable for second generation bioethanol production

Background: Robust second generation bioethanol processes require microorganisms able to ferment inhibitory lignocellullosic hydrolysates. In this study, the inhibitor tolerance and flocculation characteristics of Saccharomyces cerevisiae CCUG53310 were evaluated in comparison with S. cerevisiae CBS8066. Results: The flocculating strain CCUG53310 could rapidly ferment all hexoses in dilute acid spruce hydrolysate, while CBS8066 was strongly inhibited in this medium. In synthetic inhibitory media, CCUG53310 was more tolerant to carboxylic acids and furan aldehydes, but more sensitive than CBS8066 to phenolic compounds. Despite the higher tolerance, the increase in expression of the YAP1, ATR1 and FLR1 genes, known to confer resistance to lignocellulose-derived inhibitors, was generally smaller in CCUG53310 than in CBS8066 in inhibitory media. The flocculation of CCUG53310 was linked to the expression of FLO8, FLO10 and one or more of FLO1, FLO5 or FLO9. Flocculation depended on cell wall proteins and Ca2+ ions, but was almost unaffected by other compounds and pH values typical for lignocellulosic media. Conclusions: S. cerevisiae CCUG53310 can be characterised as being very robust, with great potential for industrial fermentation of lignocellulosic hydrolysates relatively low in phenolic inhibitors

Proteomic Analysis of the Increased Stress Tolerance of <em>Saccharomyces cerevisiae</em> Encapsulated in Liquid Core Alginate-Chitosan Capsules

<div><p><em>Saccharomyces cerevisiae</em> CBS8066 encapsulated in semi-permeable alginate or alginate-chitosan liquid core capsules have been shown to have an enhanced tolerance towards complex dilute-acid lignocellulose hydrolysates and the lignocellulose-derived inhibitor furfural, as well as towards high temperatures. The underlying molecular reasons for these effects have however not been elucidated. In this study we have investigated the response of the encapsulation on the proteome level in the yeast cells, in comparison with cells grown freely in suspension under otherwise similar conditions. The proteomic analysis was performed on whole cell protein extracts using nLC-MS/MS with TMT® labelling and 2-D DIGE. 842 and 52 proteins were identified using each method, respectively. The abundances of 213 proteins were significantly different between encapsulated and suspended cells, with good correlation between the fold change ratios obtained by the two methods for proteins identified in both. Encapsulation of the yeast caused an up-regulation of glucose-repressed proteins and of both general and starvation-specific stress responses, such as the trehalose biosynthesis pathway, and down-regulation of proteins linked to growth and protein synthesis. The encapsulation leads to a lack of nutrients for cells close to the core of the capsule due to mass transfer limitations. The triggering of the stress response may be beneficial for the cells in certain conditions, for example leading to the increased tolerance towards high temperatures and certain inhibitors.</p> </div

Heparin-binding protein as a marker of ventriculostomy related infection and central nervous system inflammation in neuro-intensive care

Objective: Diagnosis of ventriculostomy related infections (VRI) in the neuro-intensive care unit remains chal-lenging and current biomarkers lack adequate precision. The aim of this study was to explore the potential of Heparin-binding protein (HBP) in cerebrospinal fluid (CSF) as a diagnostic biomarker of VRI. Methods: All patients treated with an external ventricular drain (EVD) between January 2009 and March 2010 at Skane university hospital in Lund, Sweden, were consecutively included. CSF samples obtained during routine care were analyzed for HBP. VRI was defined as a positive bacterial microbiology test result on a CSF sample with an erythrocyte-corrected leukocyte count of &gt; 50 x 106/l. HBP levels at VRI diagnosis was compared to peak HBP levels in non-VRI controls. Results: In total, 394 CSF samples from 103 patients were analyzed for HBP. Seven patients (6.8%) fulfilled VRI criteria. Levels of HBP were significantly higher in VRI subjects (31.7 ng/mL [IQR 26.9-40.7 ng/mL]) compared to non-VRI controls (7.7 ng/mL [IQR 4.1-24.5 ng/mL]) (p = 0.024). The AUC of the receiver operating char-acteristic (ROC) curve was 0.76 (95% confidence interval [CI], 0.62-0.90). Among non-VRI patients, HBP was highest in patients with acute bacterial meningitis. Patients with subarachnoid hemorrhage displayed higher HBP levels than those with traumatic brain injury or shunt dysfunction. Conclusions: HBP levels were higher in VRI subjects and varied between patients and different diagnoses. To validate the clinical usefulness and added value of HBP as a biomarker for VRI, the results need to be confirmed in larger studies with head-to-head comparisons to current biomarkers

A novel chimaeric flocculation protein enhances flocculation in Saccharomyces cerevisiae

Yeast flocculation is the reversible formation of multicellular complexes mediated by lectin-like cell wall proteins binding to neighbouring cells. Strong flocculation can improve the inhibitor tolerance and fermentation performance of yeast cells in second generation bioethanol production. The strength of flocculation increases with the size of the flocculation protein and is strain dependent. However, the large number of internal repeats in the sequence of FLO1 from Saccharomyces cerevisiae S288c makes it difficult to recombinantly express the gene to its full length. In the search for novel flocculation genes resulting in strong flocculation, we discovered a DNA sequence, FLONF, that gives NewFlo phenotype flocculation in S. cerevisiae CEN.PK 113-7D. The nucleotide sequence of the internal repeats of FLONF differed from those of FLO1. We hypothesized that a chimaeric flocculation gene made up of a FLO1 variant derived from S. cerevisiae S288c and additional repeats from FLONF from S. cerevisiae CCUG 53310 would be more stable and easier to amplify by PCR. The constructed gene, FLOw, had 22 internal repeats compared to 18 in FLO1. Expression of FLOw in otherwise non-flocculating strains led to strong flocculation. Despite the length of the gene, the cassette containing FLOw could be easily amplified and transformed into yeast strains of different genetic background, leading to strong flocculation in all cases tested. The developed gene can be used as a self-immobilization technique or to obtain rapidly sedimenting cells for application in e.g. sequential batches without need for centrifugation.</p

<i>S. cerevisiae</i> encapsulated in alginate chitosan capsules.

<p>Capsules full of cells at the time of sampling for proteome analysis. Major unit of the ruler is in centimetres.</p

Functional categories enriched among up-regulated proteins.

<p>Enriched (p<0.01) functional categories among up-regulated proteins in encapsulated yeast, as analysed using the MIPS functional category enrichment tool (FUNCAT, <a href="http://www.helmholtz-muenchen.de/en/mips/projects/funcat" target="_blank">http://www.helmholtz-muenchen.de/en/mips/projects/funcat</a>). The number of proteins in each category is shown in parentheses.</p

Functional categories enriched among down-regulated proteins.

<p>Enriched (p<0.01) functional categories among down-regulated proteins in encapsulated yeast, as analysed using the MIPS functional category enrichment tool (FUNCAT, <a href="http://www.helmholtz-muenchen.de/en/mips/projects/funcat" target="_blank">http://www.helmholtz-muenchen.de/en/mips/projects/funcat</a>). The number of proteins in each category is shown in parentheses.</p

Fermentation profiles of encapsulated and free <i>S. cerevisiae</i>.

<p>Glucose and ethanol concentration profiles of encapsulated (◊, □) and free (▵, ○) cells during anaerobic batch cultivations.</p