Grape and wine analysis: Oenologists to exploit advanced test kits.
Charnock, S. C. & McCleary, B. V. (2005). Revue des Enology, 117, 1-5.
It is without doubt that testing plays a pivotal role throughout the whole of the vinification process. To produce the best possible quality wine and to minimise process problems such as “stuck” fermentation or troublesome infections, it is now recognised that if possible testing should begin prior to harvesting of the grapes and continue through to bottling. Traditional methods of wine analysis are often expensive, time consuming, require either elaborate equipment or specialist expertise and frequently lack accuracy. However, enzymatic bio-analysis enables the accurate measurement of the vast majority of analytes of interest to the wine maker, using just one piece of apparatus, the spectrophotometer (see previous issue No. 116 for a detailed technical review). Grape juice and wine are amenable to enzymatic testing as being liquids they are homogenous, easy to manipulate, and can generally be analysed without any sample preparation.
Megazyme “advanced” wine test kits general characteristics and validation.
Charnock, S. J., McCleary, B. V., Daverede, C. & Gallant, P. (2006). Reveue des Oenologues, 120, 1-5.
Many of the enzymatic test kits are official methods of prestigious organisations such as the Association of Official Analytical Chemicals (AOAC) and the American Association of Cereal Chemists (AACC) in response to the interest from oenologists. Megazyme decided to use its long history of enzymatic bio-analysis to make a significant contribution to the wine industry, by the development of a range of advanced enzymatic test kits. This task has now been successfully completed through the strategic and comprehensive process of identifying limitations of existing enzymatic bio-analysis test kits where they occurred, and then using advanced techniques, such as molecular biology (photo 1), to rapidly overcome them. Novel test kits have also been developed for analytes of emerging interest to the oenologist, such as yeast available nitrogen (YAN; see pages 2-3 of issue 117 article), or where previously enzymes were simply either not available, or were too expensive to employ, such as for D-mannitol analysis.
Biosynthesis of ethanol and hydrogen by glycerol fermentation using Escherichia coli.
Chaudhary, N., Ngadi, M. O., Simpson, B. K. & Kassama, L. S. (2011). Advances in Chemical Engineering and Science, 1, 83-89.
Production of high value products from glycerol via anaerobic fermentation is of utmost importance for the biodiesel industry. The microorganism Escherichia coli (E. coli) K12 was used for fermentation of glycerol. The effects of glycerol concentration and headspace conditions on the cell growth, ethanol and hydrogen production were investigated. A full factorial experimental design with 3 replicates was conducted in order to test these factors. Under the three headspace conditions tested, the increase of glycerol concentration accelerated glycerol fermentation. The yields of hydrogen and ethanol were the lowest when glycerol concentration of 10 g/L was used. The maximum production of hydrogen was observed with an initial glycerol concentration of 25 g/L at a final concentration of hydrogen was 32.15 mmol/L. This study demonstrated that hydrogen production negatively affects cell growth. Maximum ethanol yield was obtained with a glycerol concentration of 10 g/L and was up to 0.40 g/g glycerol under membrane condition headspace. Statistical optimization showed that optimal conditions for hydrogen production are 20 g/L initial glycerol with initial sparging of the reactor headspace. The optimal conditions for ethanol production are 10 g/L initial glycerol with membrane.
The proteomic response of Saccharomyces cerevisiae in very high glucose conditions with amino acid supplementation.
Pham, T. K. & Wright, P. C. (2008). Journal of Proteome Research, 7(11), 4766-4774.
Ethanol yield by Saccharomyces cerevisiae in very high glucose (VHG) media with an amino acid supplement was investigated. Amino acid supplementation led to positive cell responses, including reduced lag time and increased cell viability in VHG media. A quantitative shotgun proteomic analysis was used to understand how amino acid supplemented S. cerevisiae responds to high osmotic conditions. iTRAQ data revealed that most proteins involved in glycolysis and pentose phosphate pathways were up-regulated under high glucose shock. Reactivation of amino acid metabolism was also observed at the end of the lag phase. The relative abundance of most identified proteins, including aminoacyl-tRNA biosynthesis proteins, and heat-shock proteins, remained unchanged in the hours immediately following application of glucose shock. However, the expression of these proteins increased significantly at the end of the lag phase. Furthermore, the up-regulation of trehalose and glycogen biosynthesis proteins, first maintaining then latterly increasing glycolysis pathway activity was also observed. This was verified by enhanced ethanol yields at 10 and 12 h (0.43 and 0.45 g ethanol/g glucose) compared to 2 h (0.32 g ethanol/g glucose). These data combined with relevant metabolite measurements demonstrates that enhanced ethanol fermentation under VHG conditions can be achieved with the aid of amino acid supplementation.
Comparative transcriptomic profile analysis of fed-batch cultures expressing different recombinant proteins in Escherichia coli.
Sharma, A. K., Mahalik, S., Ghosh, C., Singh, A. B. & Mukherjee, K. J. (2011). AMB Express, 1(1), 33.
There is a need to elucidate the product specific features of the metabolic stress response of the host cell to the induction of recombinant protein synthesis. For this, the method of choice is transcriptomic profiling which provides a better insight into the changes taking place in complex global metabolic networks. The transcriptomic profiles of three fed-batch cultures expressing different proteins viz. recombinant human interferon-beta (rhIFN-β), Xylanase and Green Fluorescence Protein (GFP) were compared post induction. We observed a depression in the nutrient uptake and utilization pathways, which was common for all the three expressed proteins. Thus glycerol transporters and genes involved in ATP synthesis as well as aerobic respiration were severely down-regulated. On the other hand the amino acid uptake and biosynthesis genes were significantly repressed only when soluble proteins were expressed under different promoters, but not when the product was expressed as an inclusion body (IB). High level expression under the T7 promoter (rhIFN-β and xylanase) triggered the cellular degradation machinery like the osmoprotectants, proteases and mRNA degradation genes which were highly up-regulated, while this trend was not true with GFP expression under the comparatively weaker ara promoter. The design of a better host platform for recombinant protein production thus needs to take into account the specific nature of the cellular response to protein expression.
Proteomic analysis of Saccharomyces cerevisiae under high gravity fermentation conditions.
Pham, T. K., Chong, P. K., Gan, C. S. & Wright, P. C. (2006). Journal of Proteome Research, 5(12), 3411-3419.
Saccharomyces cerevisiae KAY446 was utilized for ethanol production, with glucose concentrations ranging from 120 g/L (normal) to 300 g/L (high). Although grown in a high glucose environment, S. cerevisiae still retained the ability to produce ethanol with a high degree of glucose utilization. iTRAQ-mediated shotgun proteomics was applied to identify relative expression change of proteins under the different glucose conditions. A total of 413 proteins were identified from three replicate, independent LC-MS/MS runs. Unsurprisingly, many proteins in the glycolysis/gluconeogenesis pathway showed significant changes in expression level. Twenty five proteins involved in amino acid metabolism decreased their expression, while the expressions of 12 heat-shock related proteins were also identified. Under high glucose conditions, ethanol was produced as a major product. However, the assimilation of glucose as well as a number of byproducts was also enhanced. Therefore, to optimize the ethanol production under very high gravity conditions, a number of pathways will need to be deactivated, while still maintaining the correct cellular redox or osmotic state. Proteomics is demonstrated here as a tool to aid in this forward metabolic engineering.
Rapid monitoring of glycerol in fermentation growth media: Facilitating crude glycerol bioprocess development.
Abad, S., Pérez, X., Planas, A. & Turon, X. (2014). Talanta, 121, 210-214.
Recently, the need for crude glycerol valorisation from the biodiesel industry has generated many studies for practical and economic applications. Amongst them, fermentations based on glycerol media for the production of high value metabolites are prominent applications. This has generated a need to develop analytical techniques which allow fast and simple glycerol monitoring during fermentation. The methodology should be fast and inexpensive to be adopted in research, as well as in industrial applications. In this study three different methods were analysed and compared: two common methodologies based on liquid chromatography and enzymatic kits, and the new method based on a DotBlot assay coupled with image analysis. The new methodology is faster and cheaper than the other conventional methods, with comparable performance. Good linearity, precision and accuracy were achieved in the lower range (10 or 15 g/L to depletion), the most common range of glycerol concentrations to monitor fermentations in terms of growth kinetics.
Repeated batch ethanolic fermentation of very high gravity medium by immobilized Saccharomyces cerevisiae.
Puligundla, P., Poludasu, R. M., Rai, J. K. & Obulam, V. S. R. (2011). Annals of Microbiology, 61(4), 863-869.
The main objective of this study was to evaluate the effect of yeast immobilization on ethanolic fermentation of very high gravity (VHG) medium and to determine the concentrations of yeast storage carbohydrates like trehalose and glycogen during the process. Repeated batch ethanolic fermentation of VHG medium was carried out using Saccharomyces cerevisiae immobilized separately within Ca-alginate and κ-carrageenan polymers. Immobilization yields (Y1) were between 80 and 90% and ethanol yields (YP/S) were more than 0.41 with both carriers. An average fermentation efficiency of nearly 70% was observed in 48-h fermentation batches. Compared to free cells, a reduction of more than 50% in the accumulated trehalose, and a two-fold increase in intracellular glycogen levels were observed in immobilized yeast cells at 24 and 48 h of fermentation, respectively, with both carriers. The increased viability (up to four-fold higher) upon 18% ethanol treatment for 2 h, and the sustained viability over four successive batches of immobilized cells showed the protective nature of the polymer carriers. The chemical nature of the carriers was not found to have any adverse effect on ethanol yields. Application of immobilized yeast in porous matrices may serve as a feasible and better technique for ethanol production, at both pilot and industrial scale.
Methane production via anaerobic digestion of glycerol: a comparison of conventional (CSTR) and high‐rate (PABR) digesters.
Vlassis, T., Stamatelatou, K., Antonopoulou, G. & Lyberatos, G. (2013). Journal of Chemical Technology and Biotechnology, 88(11), 2000-2006.
BACKGROUND: Biodiesel is an alternative to fossil fuels and can be used directly in internal combustion engines when mixed with diesel. The economic feasibility of biodiesel production necessitates the valorisation of glycerol, which is produced in large quantities (equal to 10% of the biodiesel produced). Anaerobic digestion is applicable to a variety of organic residues yielding biogas rich in methane. In order to estimate the net potential of glycerol to yield methane, pure glycerol was selected to avoid any effect from the impurities in crude glycerol.
RESULTS: The anaerobic digestion of pure glycerol was studied in two types of bioreactors: a continuous stirred tank reactor (CSTR) and a baffled reactor (periodic anaerobic baffled reactor, PABR). Both reactors were operated in mesophilic conditions (35°C) at various organic loading rates. The maximum glycerol loading achieved in a CSTR was 0.25 g COD L-1 d-1, yielding 0.074 ± 0.009 L CH4 L-1 d-1. On the other hand, PABR allowed glycerol degradation at a loading of 3 g COD L-1 d-1 yielding 0.993 ± 0.102 L CH4 L-1 d-1.
CONCLUSION: PABR was proved to be more efficient since it was subjected to a 10-fold higher organic loading rate than CSTR. Moreover, its performance was much higher in terms of COD removal and methane productivity.
Drum drying performance of condensed distillers solubles and comparison to that of physically modified condensed distillers solubles.
Milczarek, R. R. & Liu, K. (2015). Food and Bioproducts Processing, 94, 208-217.
Condensed distillers solubles (CDS) is a viscous, syrupy co-product of ethanol production from corn or other starchy grains; CDS exhibits strong recalcitrance to drying due to its chemical composition, which includes a substantial amount of glycerol. The objectives of this study were to determine the drum drying performance of CDS and to compare it to that of a physically modified CDS (MCDS) having a reduced glycerol level. Material type (CDS vs. MCDS), dwell time, drum internal steam temperature, and gap width were evaluated for their effects on the final moisture content, water activity, and color of the dried product. While both CDS and MCDS could be dried to a range of endpoint moisture contents, dried CDS exhibited a narrow range of water activity compared to that of MCDS. Gap width was found to be the predominant factor affecting dried product color. This work demonstrates that drum drying can effectively reduce the moisture content of CDS, even though the water activity of the dried product cannot be reduced beyond ∼0.45. In contrast, MCDS can be readily drum-dried into a shelf-stable, flaked product with a pleasing appearance.
Comparison of Glucose, Glycerol and Crude Glycerol Fermentation by Escherichia Coli K12.
Chaudhary, N., Ngadi, M. O. & Simpson, B. (2012). Journal of Bioprocessing & Biotechniques.
Hydrogen and ethanol production from glucose, glycerol and crude glycerol fermentation using Escherichia coli was investigated. Crude glycerol used in this study contained 80% glycerol, 2.6% ash, 12.3% moisture, 1.7% free fatty acid, 3.4% MONG (matter organic non-glycerol), 2519 mg/kg sulphur and 9000 ppm sodium. The maximum yield of ethanol from crude glycerol of 0.36 g/g, corresponding to an ethanol concentration of 3.6 g/L was obtained at 10 g/L initial glycerol concentration, 5 g/L tryptone concentration and 100 rpm mixing speed. Comparable yields were obtained at the mixing speeds of 150 and 200 rpm. On comparison, this yield corresponded to 105% of the yield (0.34 g/g) obtained from pure glycerol at the same conditions and 85% of the maximum yield (0.42 g/g) of ethanol obtained from pure glycerol at 10 g/L initial glycerol concentration, 10 g/L tryptone concentration and 200 rpm mixing speed. Additionally, Escherichia coli growth for glycerol was characterized and compared to that for glucose with 10 % substrate concentration at 37°C and 200 rpm mixing speed. The net growth rate for glucose and glycerol were 0.43 and 0.26 h-1, respectively. The maximum dry weight attained for glucose and glycerol were 0.12 and 0.04 g/L, respectively.
From crude glycerol to carotenoids by using a Rhodotorula glutinis mutant.
Cutzu, R., Coi, A., Rosso, F., Bardi, L., Ciani, M., Budroni, M., Zara, G., Zara, S. & Mannazzu, I. (2013). World Journal of Microbiology and Biotechnology, 29(6), 1009-1017.
In this work eighteen red yeasts were screened for carotenoids production on glycerol containing medium. Strain C2.5t1 of Rhodotorula glutinis, that showed the highest productivity, was UV mutagenized. Mutant 400A15, that exhibited a 280 % increase in β–carotene production in respect to the parental strain, was selected. A central composite design was applied to 400A15 to optimize carotenoids and biomass productions. Regression analyses of the quadratic polynomial equations obtained (R2 = 0.87 and 0.94, for carotenoids and biomass, respectively) suggest that the models are reliable and significant (P < 0.0001) in the prediction of carotenoids and biomass productions on the basis of the concentrations of crude glycerol, yeast extract and peptone. Accordingly, total carotenoids production achieved (14.07 ± 1.45 mg l-1) under optimized growth conditions was not statistically different from the maximal predicted (14.64 ± 1.57 mg l-1) (P < 0.05), and it was about 100 % higher than that obtained under un-optimized conditions. Therefore mutant 400A15 may represent a biocatalyst of choice for the bioconversion of crude glycerol into value-added metabolites, and a tool for the valorization of this by-product of the biodiesel industry.