Factors affecting the ethanol productivity of yeast in molasses

The ethanol production by a laboratory yeast strain, X2180-1B, was less than half that by an alcohol yeast, YOY655, in a molasses medium containing 30% sugars, although X2180-1B produced approximately the same amount of ethanol as YOY655 in a nutrition medium with the same sugar content. The weak productivity of X2180-1B in the molasses was ascribed to the limitation of sucrose hydrolysis in the molasses. The invertase activity of X2180-1B was 0.019 (mmol sucrose/min/mg protein) in the nutrition medium, but substantially zero in the molasses, while that of YOY655 was 1.75 in the nutrition medium and 1.15 even under the inhibitory conditions in molasses. External addition of invertase greatly enhanced the ethanol productivity of only X2180-1B. The inhibitory factors of invertase in molasses were heat-stable and dialyzable substances.     

Biosynthesis of invertase by Saccharomyces cerevisiae with sugarcane molasses as substrate

Biosynthesis of invertase by Saccharomyces cerevisiae 01K32 was inversely proportional to the concentration of sugarcane blackstrap molasses included in the medium. In a fermenter, an intracellular invertase activity of 440 U/g dry cells was obtained.     

Immobilized Sclerotinia sclerotiorum invertase to produce invert sugar syrup from industrial beet molasses by product

The fungus Sclerotinia sclerotiorum produces invertase activity during cultivation on many agroindustrial residues. The molasses induced invertase was purified by DEAE-cellulose chromatography. The molecular mass of the purified enzyme was estimated at 48 kDa. Optimal temperature was determined at 60 °C and thermal stability up to 65 °C. The enzyme was stable between pH 2.0 and 8.0; optimum pH was about 5.5. Apparent K_m and V_max for sucrose were estimated to be respectively 5.8 mM and 0.11 μmol/min. The invertase was activated by β-mercaptoethanol. Free enzyme exhibited 80 % of its original activity after two month’s storage at 4 °C and 50 % after 1 week at 25 °C. In order to investigate an industrial application, the enzyme was immobilized on alginate and examined for invert sugar production by molasses hydrolysis in a continuous bioreactor. The yield of immobilized invertase was about 78 % and the activity yield was 59 %. Interestingly the immobilized enzyme hydrolyzed beet molasses consuming nearly all sucrose. It retained all of its initial activity after being used for 4 cycles and about 65 % at the sixth cycle. Regarding productivity; 20 g/l of molasses by-product gave the best invert sugar production 46.21 g/day/100 g substrate related to optimal sucrose conversion of 41.6 %.     

Fungal Invertase as an Aid for Fermentation of Cane Molasses into Ethanol

Comparative studies of the fermentation of cane molasses into ethanol by Saccharomyces cerevisiae in the presence or absence of fungal invertase were performed. When cane molasses was fermented by the yeast at 30°C and pH 5.0, the presence of the enzyme had no effect on ethanol production. At pH 3.5, ethanol production was increased by the addition of invertase. At 40°C, the addition of invertase increased ethanol production by 5.5% at pH 5.0 and by 20.9% at pH 3.5.     

Ultrasound effects on invertase from Aspergillus niger

Ultrasound effects on the release and activity of invertase from Aspergillus niger cultivated in a medium containing sucrose and peptone and in another with sugar-cane molasses and peptone were investigated. Irradiation was conducted for periods of 2–10 min. with waves of amplitude 20 and 40 using an ultrasound processor of 20 kHz. Product formation was determined as reducing equivalents formed by time units using 3,5-dinitrosalicylic acid. Total and specific activities of the culture supernatants were compared in the presence and absence of sonication. Both amplitudes promoted a significant increase of total invertase activity in the time periods investigated and the highest values were obtained with an amplitude of 20. Ultrasound irradiation caused cell disruption, thus releasing invertase and, after 4 min, activation of the enzyme also occurred. The best conditions for production, extraction and activation of invertase were in molasses medium containing peptone and irradiation with ultrasound waves at 20 for 8 min. This method showed high efficiency for the extraction and activation of invertase from A. niger as well as a great potential for use in industrial processes.     

Effects of yeast invertase on ethanol production in molasses

Ethanol fermentation by an alcohol yeast, YOY655, was slower in molasses than in a nutrition rich medium with the same sugar content. Osmolality was much higher in the molasses, and the slower fermentation in the molasses was ascribed to depressed fermentation under the high osmotic pressure. Yeast invertase was an important factor in regulation the osmolality and the fermentation rate in the molasses.     

Decolorization of molasses and a dye by a newly isolated strain of the fungus Geotrichum candidum Dec 1

A fungus, Geotrichum candidum Dec 1, newly isolated as a dye-decolorizing microorganism, was used to decolorize molasses and an anthraquinone dye in shaken flasks. A degree of decolorization of molasses of 87% was achieved after 12 days of cultivation, and the maximum rate of decolorization of the dye in the culture broth was obtained in 7 days. The apparent activity of peroxidase in the molasses, which is responsible for dye decolorization, was significantly lower than that of purified peroxidase, due to the inhibition by molasses, but the inhibition was reduced after the fungus was fully grown. As two ultrafiltered fractions of molasses were similarly decolorized by Dec 1, Dec 1 apparently degraded colored substances of a wide range of molecular weights. When Dec 1 was cultivated in a medium in which sucrose in the molasses was hydrolyzed with invertase, the degree of decolorization of molasses, and rate of decolorization of the dye were similar to these obtained above.     

Invertase activity of intact cells of Saccharomyces cerevisiae growing on sugar cane molasses. I. Steady-state continuous culture tests

During the steady-state continuous culture of Saccharomyces cerevisiae on sugar cane blackstrap molasses under different experimental conditions, oscillatory variations of the invertase activity of the intact yeast cells were observed. The continuous morphological changes of the cells wall and of the periplasmic space affecting the interaction between invertase and sucrose molecules could be responsible by the observed oscillatory phenomena. The average invertase activity at the steady state is linearly correlated to the cell's growth rate.     

Nutritional composition of black soldier fly larvae feeding on agro-industrial by-products

Black soldier fly (BSF) larvae, Hermetia illucens L. (Diptera: Stratiomyidae), bio-convert organic side streams into high-quality biomass, the composition of which largely depends on the side stream used. In the present study, BSF larvae were reared on feed substrates composed of dried brewers’ spent grains, each supplemented with either water, waste brewer’s yeast, or a mixture of waste brewer’s yeast and cane molasses to obtain 12 different substrates: barley/water, barley/yeast, barley/yeast/molasses, malted barley/water, malted barley/yeast, malted barley/yeast/molasses, malted corn/water, malted corn/yeast, malted corn/yeast/molasses, sorghum-barley/water, sorghum-barley/yeast, and sorghum-barley/yeast/molasses. The crude protein, fat, ash, and mineral contents of the BSF larvae fed each feed substrate were quantified by chemical analyses. The effect of substrate, supplementation, and their interaction on crude protein, fat, and ash contents of BSF larval body composition was significant. Calcium, phosphorus, and potassium were the most abundant macrominerals in the larvae and their concentrations differed significantly among substrates. These findings provide important information to support the use of BSF larval meal as potential new source of nutrient-rich and sustainable animal feed ingredients to substitute expensive and scarce protein sources such as fishmeal and soya bean meal.     

Comparison of the hydrolytic activity of commercial cellulase preparations

Summary Two different quality types of sugar-cane molasses containing a total sugar content of 48%–50% (w/v) and 35%–42% (w/v) were investigated for Zymomonas biothanol production. Molasses concentrations of up to 250 g/l (1:3 dilution) were successfully fermented within 24 h despite a higher salt concentration in the lower grade molasses. Higher molasses concentrations (300 g/l) led to fructose accumulation. The addition of sucrose to a final sugar concentration of 15% (w/v) led to 10% (v/v) ethanol with conversion efficiencies up to 96%. Sorbitol levels were negligible, but increased up to tenfold upon addition of invertase. Offprint requests to: H. W. Doelle     

A forced flow membrane enzyme reactor for sucrose inversion using molasses

We have developed a bioreactor which uses enzyme immobilized within a ceramic membrane support (1 mm thickness). Substrate is forced through the membrane by cross-flow filtration with the reaction taking place during the process of crossing the membrane. The bioreactor is termed forced-flow membrane enzyme reactor, FFMER. Invertase, which uses sucrose to form glucose and fructose, was tested in this system. The immobilized invertase membrane converted 100% of the sucrose in a feed stream made up of a 50% molasses solution. Because molasses contains many substances besides sucrose, this method is applicable to processes using substrates present in impure feeds.     

重组大肠杆菌利用蔗糖及糖蜜发酵生产丁二酸

富含蔗糖的甘蔗糖蜜可作为制备丁二酸的廉价原料。然而生产丁二酸的潜力菌株大肠杆菌Escherichia coli AFP111不能代谢蔗糖。为了使其具有蔗糖代谢能力,将E.coli W中非PTS蔗糖利用系统蔗糖通透酶的编码基因csc B,果糖激酶的编码基因csc K和蔗糖水解酶的编码基因csc A克隆并表达到AFP111中,获得重组菌株AFP111/p MD19T-csc BKA。经厌氧发酵验证,重组菌株72 h消耗20 g/L蔗糖,丁二酸产量达到12 g/L。在3L发酵罐中采用有氧阶段培养菌体、厌氧阶段发酵的两阶段发酵方式,厌氧发酵30 h,重组菌株以蔗糖和糖蜜为碳源丁二酸产量分别为34 g/L和30 g/L。结果表明,通过外源引入非PTS蔗糖利用系统,重组菌株具有较强的代谢蔗糖生长及合成丁二酸的能力,并且能够利用廉价糖蜜发酵制备丁二酸。     

Flow injection analysis system for the supervision of industrial chromatographic downstream processing in biotechnology

Sugar beet molasses is a natural resource for various products used in daily life, ranging from sucrose to amino acids for pharmaceutical industry. The separation of molasses into these high value components is performed on a large scale by ion exchange/exclusion chromatography. A biosensor system was set up for the “in time” analysis of serine and sucrose during molasses desugarisation. -Serine was analysed with the multi-enzyme system -serine dehydratase/lactic dehydrogenase and photometric detection of the NADH consumed. Sucrose was determined with invertase/mutarotase/glucose oxidase and the oxygen consumed was monitored amperometrically. An analysis could be performed within 2–5 min by directly injecting samples from the chromatographic process into the flow injection analysis system. The determination range for the sucrose analysis was 0–2.5 gl⁻¹ and for the analysis of -serine 0–0.5 gl⁻¹. The standard deviation for the measurement of -serine was 1.7%.     

Influence of Ammonium Salts and Cane Molasses on Growth of Alcaligenes eutrophus and Production of Polyhydroxybutyrate

The production of polyhydroxybutyrate (PHB) by Alcaligenes eutrophus DSM 545 was studied in a synthetic medium with 3% glucose at pH 7.0 supplemented with several ammonium substrates and cane molasses. Growth was measured by dry cell weight, and the PHB content was measured by gas chromatography. The effects of ammonium sources such as sulfate, nitrate, phosphate, and chloride salts and those of different ammonium sulfate concentrations were evaluated. The best growth and PHB production were obtained with ammonium sulfate; however, NH(inf4)(sup+) concentrations between 0.5 and 1.5 g/liter showed no significant difference. Ammonium sulfate was therefore used as the sole source of NH(inf4)(sup+) for experiments with cane molasses as the growth activator. Optimal growth and PHB production were obtained with 0.3% molasses. However, the yields of biomass (39 to 48%) and PHB (17 to 26%) varied significantly among the different ammonium substrates and cane molasses concentrations.     

Production of ethanol by filamentous and yeast-like forms of <Emphasis Type="Italic">Mucor indicus</Emphasis> from fructose,glucose, sucrose,and molasses

The fungus Mucor indicus is found in this study able to consume glucose and fructose, but not sucrose in fermentation of sugarcane and sugar beet molasses. This might be an advantage in industries which want to selectively remove glucose and fructose for crystallisation of sucrose present in the molasses. On the other hand, the fungus assimilated sucrose after hydrolysis by the enzyme invertase. The fungus efficiently grew on glucose and fructose and produced ethanol in synthetic media or from molasses. The cultivations were carried out aerobically and anaerobically, and manipulated toward filamentous or yeast-like morphology. Ethanol was the major metabolite in all the experiments. The ethanol yield in anaerobic cultivations was between 0.35 and 0.48 g/g sugars consumed, depending on the carbon source and the growth morphology, while a yield of as low as 0.16 g/g was obtained during aerobic cultivation. The yeast-like form of the fungus showed faster ethanol production with an average productivity of 0.90 g/l h from glucose, fructose and inverted sucrose, than the filamentous form with an average productivity of 0.33 g/l h. The biomass of the fungus was also analyzed with respect to alkali-insoluble material (AIM), chitin, and chitosan. The biomass of the fungus contained per g maximum 0.217 g AIM and 0.042 g chitosan in yeast-like cultivation under aerobic conditions.     

Technology and economics of ethanol production from fodder beets via solid-phase fermentation

Summary Operating conditions for our semi-continuous, solid-phase fermentation system were optimized for conversion of fodder beets to fuel ethanol and distiller's wet feed (DWF). This information was then used to estimate operating parameters achievable in a commercial plant, and likely baseline production costs of such a plant. Initial acidification of pulp to pH 2.9–3.2 was effective in controlling bacterial contamination. The maximum operating capacity of the fermentor was approximately 92%, with 75% used for commercial application. A fermentation time of 24 h was sufficient to completely ferment the beet pulp to 8–9% (v/v) ethanol. Based on these parameters, a fodder beet cost of $19.25/metric ton ($17.50/ton), other operating and capital costs, and a PF credit of $0.14/L ($0.53/gal), ethanol production costs were estimated to be $0.49/L ($1.87/gal).     

Invertase activity of intact cells of Saccharomyces cerevisiae growing on sugarcane molasses. II. Unsteady-state continuous-culture tests

During unsteady-state continuous culture of Saccharomyces cerevisiae on sugarcane blackstrap molasses, the invertase activity of the intact yeast cells oscillated. Disturbances were produced by changing medium composition, air rate, impeller speed, and dilution rate. The influence of the oxygen supply rate and of the dilution rate on the invertase activity depend on the medium composition. The highest invertase activity was obtained when, after a steady-state attained using unsupplemented culture medium, nutrients were added to the feeding mash. A Monod-like equation seems to be the best representation of the correlation between the specific rate of reducing sugars consumption and the specific rate of nonreducing sugar hydrolysis by the yeast cells.     

Use of complex media for the production of scleroglucan by Sclerotium rolfsii MTCC 2156

Submerged fermentation was carried out for the production of scleroglucan by Sclerotium rolfsii MTCC 2156 using complex media, such as coconut water, sugarcane molasses and sugarcane juice at 28+/-2 degrees C and 180 rpm for 72 h. Sugarcane juice gave maximum scleroglucan production of 23.87 g/l as compared to 12.58 and 18.45 g/l with coconut water and sugarcane molasses, respectively. Utilization of these substrates would be ecologically sound and economically advantageous.     

Ethanol tolerance, sugar tolerance and invertase activities of some yeast strains isolated from steep water of fermenting cassava tubers

E kunsanmi , T.J. & O dunfa , S.A. 1990. Ethanol tolerance, sugar tolerance and invertase activities of some yeast strains isolated from steep water of fermenting cassava tubers. Journal of Applied Bacteriology 69 , 672–675.
Thirteen yeasts isolated from the steep water of fermenting cassava tubers were screened for ethanol tolerance. Three strains which showed measurable growth in medium containing 10% (v/v) ethanol were also sugar-tolerant and grew well in medium containing 25% (w/v) glucose. One of the strains, YC3, was found to possess much higher invertase activity than the other two and could be of value in ethanol production from molasses. Further search for industrially useful yeasts in African fermented foods is suggested.     

Requirements of cleavage of high mannose oligosaccharides in glycoproteins by peptide N-glycosidase F

Peptide N-glycosidase from Flavobacterium meningosepticum cleaves complex as well as neutral glycoproteins (Plummer, T.H., Jr., Elder, J.H., Alexander, S., Phelan, A.W., and Tarentino, A.L. (1984) J. Biol. Chem. 259, 10700-10704). Examples of neutral glycoprotein substrates include ribonuclease B (one high mannose oligosaccharide chain) and yeast external invertase (nine chains/invertase subunit). The rate of deglycosylation by the glycosidase was greatly enhanced if the glycoprotein substrate was denatured prior to enzyme treatment, from a low of 11-fold for external invertase to a high of 844-fold for ribonuclease B. Peptide N-glycosidase F was unable to cleave the asparaginyl-N-acetylglucosamine bond in endo-beta-N-acetylglucosaminidase H-modified external invertase or ribonuclease B, although that in similarly modified glycopeptide substrate was cleaved. Ribonuclease B was digested sequentially with various exoglycosidases to produce an oligosaccharide chain of varied length. Using the resulting forms of ribonuclease B as substrates for peptide N-glycosidase F, the minimum oligosaccharide chain for cleavage was the di-N-acetyl-chitobiosyl core unit.