Production Of Higher Alcohols Research Articles

Production of 2-methyl-1-butanol in engineered Escherichia coli

Recent progress has been made in the production of higher alcohols by harnessing the power of natural amino acid biosynthetic pathways. Here, we describe the first strain of Escherichia coli developed to produce the higher alcohol and potential new biofuel 2-methyl-1-butanol (2MB). To accomplish this, we explored the biodiversity of enzymes catalyzing key parts of the isoleucine biosynthetic pathway, finding that AHAS II (ilvGM) from Salmonella typhimurium and threonine deaminase (ilvA) from Corynebacterium glutamicum improve 2MB production the most. Overexpression of the native threonine biosynthetic operon (thrABC) on plasmid without the native transcription regulation also improved 2MB production in E. coli. Finally, we knocked out competing pathways upstream of threonine production (ΔmetA, Δtdh) to increase its availability for further improvement of 2MB production. This work led to a strain of E. coli that produces 1.25 g/L 2MB in 24 h, a total alcohol content of 3 g/L, and with yields of up to 0.17 g 2MB/g glucose.

Metabolic engineering of Escherichia coli for 1-butanol and 1-propanol production via the keto-acid pathways

Production of higher alcohols via the keto-acid intermediates found in microorganism's native amino-acid pathways has recently shown promising results. In this work, an Escherichia coli strain that produces 1-butanol and 1-propanol from glucose was constructed. The strain first converts glucose to 2-ketobutyrate, a common keto-acid intermediate for isoleucine biosynthesis. Then, 2-ketobutyrate is converted to 1-propanol through reactions catalyzed by the heterologous decarboxylase and dehydrogenase, or to 1-butanol via the chemistry involved in the synthesis of the unnatural amino acid norvaline. We systematically improved the synthesis of 1-propanol and 1-butanol through deregulation of amino-acid biosynthesis and elimination of competing pathways. The final strain demonstrated a production titer of 2 g/L with nearly 1:1 ratio of butanol and propanol.

Comparative study of batch and continuous multi-stage fixed-bed tower (MFBT) bioreactor during wine-making using freeze-dried immobilized cells

A freeze-dried immobilized biocatalyst produced by immobilization of Saccharomyces cerevisiae AXAZ-1 yeast cells on gluten pellets and subsequent freeze-drying was used in a multistage fixed-bed tower (MFBT) bioreactor for batch and continuous wine-making. The MFBT bioreactor resulted in higher alcohol productivity compared to fermentations carried out in a packed bed (PB) bioreactor and showed an important operational stability and no decrease in activity, even at low fermentation temperature (5 °C) and after storage for 6 months at 4 °C. The production of amyl alcohols proved to be temperature dependent and was significantly reduced at low temperatures. Re-activation of the freeze-dried immobilized cells after storage for 6 months resulted in further decreased content of amyl alcohols. The SPME GC/MS analysis of volatile compounds revealed no significant differences in the wines produced by MFBT and PB bioreactors, while the preliminary sensory evaluation ascertained the overall improved quality of the produced wines. Potential industrial application of MFBT bioreactor is also assessed and discussed.

Heavy sulphur compounds, higher alcohols and esters production profile of Hanseniaspora uvarum and Hanseniaspora guilliermondii grown as pure and mixed cultures in grape must

Hanseniaspora guilliermondii and Hanseniaspora uvarum were tested in grape must fermentations as pure and mixed starter cultures with Saccharomyces cerevisiae. In pure cultures, the specific growth rates found were 0.29 h − 1 for H. uvarum, 0.23 h − 1 for H. guilliermondii and 0.18 h − 1 for S. cerevisiae. No significant differences were observed between these values and those obtained in mixed cultures. Results presented in this work show that growth of apiculate yeasts during the first days of fermentation enhances the production of desirable compounds, such as esters, and may not have a negative influence on the production of higher alcohols and undesirable heavy sulphur compounds. Growth of apiculate yeasts reduced the total content of higher alcohols in wines, when compared to those produced by a pure culture of S. cerevisiae. Furthermore, the highest levels of 2-phenylethyl acetate were obtained when H. guilliermondii was inoculated in grape musts, whereas H. uvarum increased the isoamyl acetate content of wines. Apiculate yeasts produced high amounts of ethyl acetate; however, the level of this compound decreased in mixed cultures of apiculate yeasts and S. cerevisiae. When S. cerevisiae was used as a starter culture, wines showed higher concentrations of glycerol, 2-phenylethanol and ethyl hexanoate. In mixed cultures of apiculate yeasts and S. cerevisiae, wines presented amounts of methionol, acetic acid-3-(methylthio)propyl ester, 4-(methylthio)-1-butanol, 2-mercaptoethanol and cis-2-methyltetrahydro-thiophen-3-ol similar to those produced by a pure culture of S. cerevisiae. An increase in the amounts of 3-(ethylthio)-1-propanol, trans-2-methyltetrahydro-thiophen-3-ol and 3-mercapto-1-propanol was obtained in wines produced from mixed cultures with H. guilliermondii.

Co-Cu Metal Alloys from LaCo1-xCuxO3 Perovskites as Catalysts for Higher Alcohol Synthesis from Syngas

The specific surface area of ground perovskites is strongly dependent on the synthesis and pretreatment conditions. Calcining the ground LaCo1-xCuxO3-? perovskites at 5000C causes a decrease of their specific surface, but the further reductive pretreatment of the calcined sample in hydrogen leads to only a minor change in the specific surface area.The introduction of extra- and intra-perovskite lattice copper has different effects on the structure stability, reducibility and catalytic properties of LaCoO3 perovskites. Under the same pretreatment conditions the basic structure of both LaCoO3 and Cu2O/LaCoO3 samples is retained at the reduction temperature of 5000C (90 min) while that of Co-Cu based perovskites is completely collapsed producing a high dispersion of bimetallic metals and Co-Cu alloys supported on amorphous La2O3. For a series of LaCo1-xCuxO3 samples, increasing the copper content in the perovskite structure leads to an increase in the metal surface area of the reduced phase. The pretreated catalysts were tested for CO dissociation and for alcohol synthesis from syngas. The results indicated that the copper sites neighboring with cobalt atoms in the reduced perovskites decrease the activity in CO dissociation and increase the rate of higher alcohol synthesis while extra-perovkite lattice copper tends to produce methanol, methane and CO2. The overall activity in syngas conversion and higher alcohol productivity are proportional to the Co-Cu metal surface. The role of dual sites of Co and Cu for the synthesis of higher alcohols is discussed. The catalytic activity and alcohol productivity as well as product distribution depend strongly on reaction temperature and pretreatment conditions. The highest productivity of alcohols in the present study is about 73 mg/gcat/h for sample LaCo0.7Cu0.3O3.

The influence of nitrogen and biotin interactions on the performance of Saccharomyces in alcoholic fermentations

To study the impact of assimilable nitrogen, biotin and their interaction on growth, fermentation rate and volatile formation by Saccharomyces. Fermentations of synthetic grape juice media were conducted in a factorial design with yeast assimilable nitrogen (YAN) (60 or 250 mg l(-1)) and biotin (0, 1 or 10 microg l(-1)) as variables. All media contained 240 g l(-1) glucose + fructose (1 : 1) and were fermented using biotin-depleted Saccharomyces cerevisiae strains EC1118 or UCD 522. Both strains exhibited weak growth and sluggish fermentation rates without biotin. Increased nitrogen concentration resulted in higher maximum fermentation rates, while adjusting biotin from 1 to 10 microg l(-1) had no effect. Nitrogen x biotin interactions influenced fermentation time, production of higher alcohols and hydrogen sulfide (H(2)S). Maximum H(2)S production occurred in the medium containing 60 mg l(-1) YAN and 1 microg l(-1) biotin. Nitrogen x biotin interactions affect fermentation time and volatile production by Saccharomyces depending on strain. Biotin concentrations sufficient to complete fermentation may affect the organoleptic impact of wine. This study demonstrates the necessity to consider nutrient interactions when diagnosing problem fermentations.

Feasibility of ethanol production from cultivated sugarcane varieties of punjab

Alcohol production potential of six sugarcane varieties of different maturity groups (CoJ 83, CoJ 85, CoJ 82, CoJ 84, CoJ 88 and Col 148) were evaluated in both the plant and ratoon crops at PAU, SRS, Jalandhar, India. CoJ 88 was found to be the best variety having best combination of juice quality (CCS % = 13.42 & 13.85 and alcohol production L/t cane=67.28 & 69.10 ( values of alcohol production from juice are 27 % less than the actual potential of varieties which accounted for processing losses in factory) and yield (85.85 & 66.35 t/ha) which resulted in high potential of sugar and alcohol production per unit area (CCS = 11.52 & 9.19 t/ha and alcohol production = 57.76 & 45.85 hecto L/ha) in plant and ratoon crops respectively. It had good potential for alcohol production throughout the crushing season. On the basis of these studies, it was also concluded that contribution of sucrose content was much higher as compared to reducing sugars towards alcohol production so the strategy for selection of genotypes at various development stages of varieties for high alcohol production would remain same as for high sucrose (white sugar) production.

The effect of increased branched-chain amino acid transaminase activity in yeast on the production of higher alcohols and on the flavour profiles of wine and distillates

In Saccharomyces cerevisiae, branched-chain amino acid transaminases (BCAATases) are encoded by the BAT1 and BAT2 genes. BCAATases catalyse the transfer of amino groups between those amino acids and alpha-keto-acids. alpha-Keto-acids are precursors for the biosynthesis of higher alcohols, which significantly influence the aroma and flavour of yeast-derived fermentation products. The objective of this study was to investigate the influence of BAT-gene expression on general yeast physiology, on aroma and flavour compound formation and on the sensory characteristics of wines and distillates. For this purpose, the genes were overexpressed and deleted in a laboratory strain, BY4742, and overexpressed in an industrial wine yeast strain, VIN13. The data show that, with the exception of a slow growth phenotype observed for the BAT1 deletion strain, the fermentation behaviour of the strains was unaffected by the modifications. The chemical and sensory analysis of fermentation products revealed a strong correction between BAT gene expression and the formation of many aroma compounds. The data suggest that the adjustment of BAT gene expression could play an important role in assisting winemakers in their endeavour to produce wines with specific flavour profiles.

Higher alcohols concentration and its relation with the biological aging evolution

The production of 1-propanol, isobutanol, 2-methyl-1-butanol, 3-methyl-1-butanol and 2-phenylethanol by two flor yeast strains during accelerated biological aging of sherry wines with different aging time has been evaluated. The S. cerevisiae G1 strain has shown a higher production of higher alcohols than the S. bayanus F12 strain highlighting the 1-propanol production in the youngest wine. Differences between the two studied yeasts decreased in relation with the aging time of the initial wine used. The multiple regression analysis carried out to relate the higher alcohols concentration and the values obtained from an aging equation showed R-squared values ranging from 81.1 to 96.5%. The higher alcohols that best described such relation were 1-propanol, isobutanol and 3-methyl-1-butanol.

The vinification of partially dried grapes: a comparative fermentation study of Saccharomyces cerevisiae strains under high sugar stress

The study of the fermentation performance of Saccharomyces cerevisiae strains under high sugar stress during the vinification of partially dried grapes. Microvinification of partially dried grape must with sugar concentration of 35 degrees Brix was performed using four commercial strains to carry out alcoholic fermentation. A traditional red vinification without nutrients addition was applied. Yeasts displayed different efficiency to convert sugar in ethanol and varied in glycerol yield. Sugar consumption and ethanol level were attested at 80-87% and 143.5-158.0 g l(-1) respectively. High correlation between sugar and assimilable nitrogen consumption rate was observed. Statistical treatment of data by principal component analysis highlighted the different behaviours that strains exhibited in regard to the production of higher alcohols and other compounds important to wine quality. Saccharomyces cerevisiae strains displayed appreciable capability to overcome osmotic stress and to yield ethanol fermenting high sugar concentration grape must in winemaking condition. The results provided insights on the strain contribution to wine quality subordinate to stress condition. This investigation is of applicative interest for winemaking and processing industry that use high sugar concentration musts.

Studies On Fermentation, Alcohol Production And Viability In Induced Mutants Of The Brewing Yeast <I>Saccharomyces cerevisiae</I>

Brewing yeast, Saccharomyces. Cerevisiae cultured on malt extract agar was UV-irradiated. Eighteen mutant yeasts (SCM 01 – SCM 18) selected (after visual examination) were tested for fermentation, alcohol production and viability by acid and gas production, reduction in specific gravity and turbidity respectively, with sugars and delignified sugarcane bagasse. The yeasts had varied fermentation profiles in glucose, sucrose, galactose, dextrose and mannitol but did no ferment lactose. Glucose, sucrose and galactose were fermented strongly with acid and gas production. Dextrose was fermented weakly by SCM 01, SCM 06, SCM 09 and SCM 10 (with acid production only) but strongly by all other yeasts. The wild type yeast did not ferment mannitol but SCM 04 did so strongly and SCM 02, SCM 03, SCM 06, SCM 07, SCM 09, SCM 10 and SCM 13 did so weakly. Specific gravity fell sharply within 36h after inoculation and slightly thereafter. Alcohol production varied among the yeasts. Some mutants produced more alcohol than the wild type yeast. Viability was generally lower in the mutants than in the wild-type yeast in the sucrose medium. The reverse was true in the sugarcane bagasse medium. Yeasts with high viability tended to have high alcohol production ability in the sucrose medium and vice-versa. KEY WORDS: Alcohol production; fermentation; induced mutants; Saccharomyces cerevisiae; viability. Global Journal of Pure and Applied Sciences Vol.11(1) 2005: 1-7

Characterization and reactivity of Ru/single oxides catalysts for the syngas reaction

A series of ruthenium-supported catalysts were prepared and characterised to determine the effect of support interaction in the production of higher alcohol from syngas. Single oxides with various degrees of acidity/reducibility such as MoO 3, ZrO 2, WO 3 and TiO 2 were used as supports. Al 2O 3 was used as reference. XPS and TGA results indicated that, on the studied supports, partial reduction to various extents was attained. The reduction of these supports was very different: while no reduction was observed for Al 2O 3, TiO 2 and ZrO 2, WO 3 was little reduced. MoO 3 being the most reduced support. Catalytic tests indicate that the selectivity towards oxygenates seems to be linked to the reducibility of the support, since with non-easily reducible oxides (Al 2O 3, ZrO 2 and TiO 2) the alcohol production was very low, while higher selectivity was obtained on WO 3 and especially on MoO 3, the reducible oxides. A SMSI effect is invoked.

Higher-alcohols biorefinery: improvement of catalyst for ethanol conversion.

The concept of a biorefinery for higher-alcohol production is to integrate ethanol and methanol formation via fermentation and biomass gasification, respectively, with conversion of these simple alcohol intermediates into higher alcohols via the Guerbet reaction. 1-Butanol results from the self-condensation of ethanol in this multistep reaction occurring on a single catalytic bed. Combining methanol with ethanol gives a mixture of propanol, isobutanol, and 2-methyl-1-butanol. All of these higher alcohols are useful as solvents, chemical intermediates, and fuel additives and, consequently, have higher market values than the simple alcohol intermediates. Several new catalysts for the condensation of ethanol and alcohol mixtures to higher alcohols were designed and tested under a variety of conditions. Reactions of methanol-ethanol mixtures gave as high as 100% conversion of the ethanol to form high yields of isobutanol with smaller amounts of 1-propanol, the amounts in the mixture depending on the starting mixture. The most successful catalysts are multifunctional with basic and hydrogen transfer components.

The influence of clay on K 2CO 3/Co–MoS 2 catalyst in the production of higher alcohol fuel

The K 2CO 3/Co–MoS 2/clay catalyst was synthesized and its productivity and selectivity toward higher alcohol synthesis (HAS) fuel were tested at temperature and pressure ranges of 290–320 °C, H 2/CO=1.1 syngas, GHSV avg=1800 h −1, and 13 790 kPa, respectively. Highest oxygenates productivity (0.32 kg/kg catalyst/h) was obtained at 310 °C with an oxygenates selectivity of 70%. Under the above conditions, the major oxygenates produced were ethanol, 1-propanol, methanol, and isobutanol, respectively. Increasing the reaction temperature for K 2CO 3/Co–MoS 2/clay catalyst led to a decreasing trend in the selectivity of oxygenates. However, at higher temperatures, the clay-combined catalyst produced more hydrocarbons and CO 2. The results indicated that clay had a significant impact on higher alcohol synthesis when K 2CO 3 was incorporated into the Co–MoS 2, and acted as a modifier of the catalyst serving to increase the activity and selectivity to higher alcohol fuel in the temperature range of 290–310 °C.

Relations between elevated temperatures and fermentation behaviour of Kloeckera apiculata and Saccharomyces cerevisiae associated with winemaking in mixed cultures

One of the important factors affecting wine fermentation is temperature. The influence of elevated temperatures from 10 to 25 °C at 5 °C intervals on yeast growth and fermentation products were studied in mixed cultures of Kloeckera apiculata and Saccharomyces cerevisiae in grape juice. In the experiments carried out at 10 and 15 °C, K. apiculata grew and survived longer compared to trials conducted above 20 °C. In most cases, higher temperatures stimulated the production of higher alcohols but lowered the formation of esters and acetaldehyde.

Higher alcohols and esters production by Saccharomyces cerevisiae. Influence of the initial oxygenation of the grape must

Saccharomyces cerevisiae, races cerevisiae and capensis were used to carry out fermentations of grape musts with and without initial oxygen, in order to study changes in the production of higher alcohols and esters. After fermentation of the musts without initial oxygen, both yeast races produced lesser concentrations of the earlier-mentioned compounds than those obtained for the musts fermented with initial oxygenation. However a better ratio between these two types of compounds was achieved in the must without initial oxygen, so a treatment eliminating the oxygen of the must could be used to improve the sensory properties of the resulting wine.

Control of Higher Alcohol Production by Manipulation of the BAP2 Gene in Brewing Yeast

We studied whether the production of higher alcohols during wort fermentation is responsive to branched-chain amino acid metabolism. Addition of valine, isoleucine, and leucine to the brewing wort resulted in increased assimilation of each amino acid by yeast cells and caused increased production of the corresponding higher alcohol, suggesting that production of a higher alcohol is correlated with the assimilation of the corresponding amino acid. Therefore, we attempted the constitutive expression of the gene (BAP2) for the branched-chain amino acid permease in brewing yeast and investigated the effects on assimilation of branched-chain amino acids and production of higher alcohols during wort fermentation. Constitutive expression of the BAP2 gene resulted in accelerated rates of assimilation for leucine, valine, and isoleucine. This caused increased production of isoamyl alcohol derived from leucine, while an increase of isobutyl alcohol derived from valine or active amyl alcohol from isoleucine was not observed. These results suggest that there are distinct but interrelated mechanisms for the production of each higher alcohol.

Characterization of Kloeckera apiculata strains from the Friuli regionin Northern Italy

Forty-nine strains of Kloeckera apiculata, isolated from the Friuli region in Italy, were differentiated on the basis of fermentation behaviour and production of secondary compounds in two different grape musts at 18 °C. The isolates exhibited a controlled production of acetic acid, only in a few cases more that 1 g/l. In Moscato grape must the strains exhibited a more uniform behaviour for the production of higher alcohols, ethyl acetate and acetoin than in red grapes. In general, higher levels of ethanol, glycerol and acetic acid were produced in red grape must fermentation. Apiculate strains behaved differently in the two musts, with different metabolic phenotypes dominating the fermentation process. The existence of different metabolic phenotypes correlated with the must composition underlines the need to perform a selection of indigenous apiculate yeasts to obtain the desired consistent products.

High alcohol production by repeated batch fermentation using an immobilized osmotolerant Saccharomyces cerevisiae

A repeated batch fermentation system was used to produce ethanol using an osmotolerant Saccharomyces cerevisiae (VS3) immobilized in calcium alginate beads. For comparison free cells were also used to produce ethanol by repeated batch fermentation. Fermentation was carried for six cycles with 125, 250 or 500 beads using 150, 200 or 250 g glucose L−1 at 30°C. The maximum amount of ethanol produced by immobilized VS3 using 150 g L−1 glucose was only 44 g L−1 after 48 h, while the amount of ethanol produced by free cells in the first cycle was 72 g L−1. However in subsequent fed batch cultures more ethanol was produced by immobilized cells compared to free cells. The amount of ethanol produced by free cells decreased from 72 g L−1 to 25 g L−1 after the fourth cycle, while that of immobilized cells increased from 44 to 72 g L−1. The maximum amount of ethanol produced by immobilized VS3 cells using 150, 200 and 250 g glucose L−1 was 72.5, 93 and 87 g ethanol L−1 at 30°C. Journal of Industrial Microbiology & Biotechnology (2000) 24, 222–226.

Effect of threonine, cystathionine, and the branched-chain amino acids on the metabolism of Zygosaccharomyces rouxii☆

Zygosaccharomyces rouxii is an important yeast in the formation of flavor in soy sauce. In this study, we investigated the separate effects of exogenous threonine, cystathionine, and the branched-chain amino acids on the metabolism of Z. rouxii. The addition of these amino acids had significant effects on both Z. rouxii growth and glycerol and higher alcohol production. It also seemed that Z. rouxii displayed the Crabtree effect, which was independent of the added amino acids. Furthermore, we investigated the regulation of the metabolism of α-ketobutyrate, which is a key-intermediate in Z. rouxii amino acid metabolism. Threonine and cystathionine were introduced separately to stimulate the formation rate of α-ketobutyrate and the branched-chain amino acids to inhibit its conversion rate. Enzyme activities showed that these amino acids had a significant effect on the formation and conversion rate of α-ketobutyrate but that the α-ketobutyrate pool size in Z. rouxii was in balance all the time. The latter was confirmed by the absence of α-ketobutyrate accumulation.