Fermentation pattern ofZymomonas mobilis strains on different substrates—a comparative study

The optimum conditions (pH and initial sugar concentration) of fermentation for the production of ethanol by 4 strains ofZymomonas mobilis (ATCC 10988, ATCC 12526, NRRL B 4286 and IFO 13756) were studied. An initial sugar concentration of 15 % (w/v) at pH 7.0 was found to be optimal for the first two strains and 20 % (w/v) initial sugar at pH 7.0 was found to be optimal for the last two strains. The fermentation pattern of these strains on synthetic medium, cane juice and molasses were compared. Strain NRRL B 4286 showed maximum ethanol production on synthetic medium while on cane juice ATCC 10988 and ATCC 12526 performed well. However, all the strains fermented molasses poorly.     

Fermentation of Cane Molasses by Yeasts after Preservation under Conditions of High Sugar Concentration

S ummary : Fermentations of cane molasses solutions by sugar-tolerant flocculating strains of Saccharomyces cerevisiae Sa. 28 and Sacch. carlsbergensis Sa. 23, produced compact sediments from which supernatant liquid could be decanted easily. Immediate treatment of the residual yeast with molasses containing 70·2% w/v total sugars (64° Brix) at 20°, engendered viabilities of 21·9–58% and 0·043–0·25% after 3 and 100 days, respectively. Dilution of the molasses caused culture recovery with fermentation, the initial rate of which could be correlated with the % viability of the yeast culture. The technique of preserving yeast cultures by use of concentrated sugar solutions warrants further investigation and could perhaps find application in distillery practice.     

Ethanol production by thermotolerant yeast and its UV resistant mutants.

Six thermotolerant yeasts were isolated at 37 degrees C from over-ripe grapes by serial dilution technique using glucose yeast extract medium. Purified yeast cultures were screened for ethanol production at 37 degrees C by batch fermentation, using cane molasses containing 20% sugars. Sugar conversion efficiency of these isolates varied from 66.0 to 88.5% and ethanol productivity from 1.11 to 1.73 ml/l/h. The highest ethanol producing isolate was exposed to UV radiations and 13 mutants were picked up from the UV treatment exhibiting 0.1 to 1.0%, survival. The UV mutants varied in cell size from parent as well as among themselves. Determination of ethanol produced by all the mutants revealed that only five mutants resulted in 4.5 to 6.2% increase in sugar conversion and 8.25 to 18.56% increase in ethanol concentration coupled with maximum ethanol productivity of 2.4 ml/l/h in 48 h of batch fermentation of cane molasses (20% sugars) at 37 degrees C temperature.     

Simultaneous saccharification and fermentation of lignocellulosic wastes to ethanol using a thermotolerant yeast

Simultaneous saccharification and fermentation (SSF) studies were carried out to produce ethanol from lignocellulosic wastes (sugar cane leaves and Antigonum leptopus leaves) using Trichoderma reesei cellulase and yeast cells. The ability of a thermotolerant yeast, Kluyveromyces fragilis NCIM 3358, was compared with Saccharomyces cerevisiae NRRL-Y-132. K. fragilis was found to perform better in the SSF process and result in high yields of ethanol (2.5-3.5% w/v) compared to S. cerevisiae (2.0-2.5% w/v). Increased ethanol yields were obtained when the cellulase was supplemented with beta-glucosidase. The conversions with K. fragilis were completed in a short time. The substrates were in the following order in terms of fast conversions: Solka floc > A. leptopus > sugar cane.     

Yeast selection for fuel ethanol production in Brazil

Brazil is one of the largest ethanol biofuel producers and exporters in the world and its production has increased steadily during the last three decades. The increasing efficiency of Brazilian ethanol plants has been evident due to the many technological contributions. As far as yeast is concerned, few publications are available regarding the industrial fermentation processes in Brazil. The present paper reports on a yeast selection program performed during the last 12 years aimed at selecting Saccharomyces cerevisiae strains suitable for fermentation of sugar cane substrates (cane juice and molasses) with cell recycle, as it is conducted in Brazilian bioethanol plants. As a result, some evidence is presented showing the positive impact of selected yeast strains in increasing ethanol yield and reducing production costs, due to their higher fermentation performance (high ethanol yield, reduced glycerol and foam formation, maintenance of high viability during recycling and very high implantation capability into industrial fermenters). Results also suggest that the great yeast biodiversity found in distillery environments could be an important source of strains. This is because during yeast cell recycling, selective pressure (an adaptive evolution) is imposed on cells, leading to strains with higher tolerance to the stressful conditions of the industrial fermentation.     

The use of tamarind waste to improve ethanol production from cane molasses

Tamarind wastes such as tamarind husk, pulp, seeds, fruit and the effluent generated during tartaric acid extraction were used as supplements to evaluate their effects on alcohol production from cane molasses using yeast cultures. Small amounts of these additives enhanced the rate of ethanol production in batch fermentations. Tamarind fruit increased ethanol production (9.7%, w/v) from 22.5% reducing sugars of molasses as compared to 6.5% (w/v) in control experiments lacking supplements after 72 h of fermentation. In general, the addition of tamarind supplements to the fermentation medium showed more than 40% improvement in ethanol production using higher cane molasses sugar concentrations. The direct fermentation of aqueous tamarind effluent also yielded 3.25% (w/v) ethanol, suggesting its possible use as a diluent in molasses fermentations. This is the first report, to our knowledge, in which tamarind-based waste products were used in ethanol production. Received 2 April 1998/ Accepted in revised form 13 November 1998     

Production of fuel ethanol at high temperature from sugar cane juice by a newly isolated Kluyveromyces marxianus

Kluyveromyces marxianus DMKU 3-1042, isolated by an enrichment technique in a sugar cane juice medium supplemented with 4% (w/v) ethanol at 35 degrees C, produced high concentrations of ethanol at both 40 and 45 degrees C. Ethanol production by this strain in shaking flask cultivation in sugar cane juice media at 37 degrees C was highest in a medium containing 22% total sugars, 0.05% (NH(4))(2)SO(4), 0.05% KH(2)PO(4), and 0.15% MgSO(4).7H(2)O and having a pH of 5.0; the ethanol concentration reached 8.7% (w/v), productivity 1.45 g/l/h and yield 77.5% of theoretical yield. At 40 degrees C, a maximal ethanol concentration of 6.78% (w/v), a productivity of 1.13 and a yield 60.4% of theoretical yield were obtained from the same medium, except that the pH was adjusted to 5.5. In a study on ethanol production in a 5l jar fermenter with an agitation speed of 300 rpm and an aeration rate of 0.2 vvm throughout the fermentation, K. marxianus DMKU 3-1042 yielded a final ethanol concentration of 6.43% (w/v), a productivity of 1.3g/l/h and a yield of 57.1% of theoretical yield.     

High-Efficiency Carbohydrate Fermentation to Ethanol at Temperatures above 40 degrees C by Kluyveromyces marxianus var. marxianus Isolated from Sugar Mills

A number of yeast strains, isolated from sugar cane mills and identified as strains of Kluyveromyces marxianus var. marxianus, were examined for their ability to ferment glucose and cane syrup to ethanol at high temperatures. Several strains were capable of rapid fermentation at temperatures up to 47 degrees C. At 43 degrees C, >6% (wt/vol) ethanol was produced after 12 to 14 h of fermentation, concurrent with retention of high cell viability (>80%). Although the type strain (CBS 712) of K. marxianus var. marxianus produced up to 6% (wt/vol) ethanol at 43 degrees C, cell viability was low, 30 to 50%, and the fermentation time was 24 to 30 h. On the basis of currently available strains, we suggest that it may be possible by genetic engineering to construct yeasts capable of fermenting carbohydrates at temperatures close to 50 degrees C to produce 10 to 15% (wt/vol) ethanol in 12 to 18 h with retention of cell viability.     

Development and characterization of butanol — Resistant strain ofClostridium acetobutylicum in molasses medium

The production of acetone—butanol—ethanol solvents from cane molasses by locally isolated culture ofClostridium acetobutylicum was limited by butanol toxicity (1.6 mol/L). The butanol tolerance of the isolated culture was increased up to 4.8 mol/L by a serial enrichment method. The butanol-resistant strain had greater efficiency for the conversion of saccharides to mixed solvents and produced 52% more butanol at the expense of acetone and ethanol than the original strain. Moreover, the fermentation profile of parent and butanol-resistant strains in anerobic fermentation of cane molasses demonstrated the superiority of the latter in terms of growth rate, time of onset of butanol production, sugar utilization, final butanol concentration and other parameters.     

Evaluation of thermotolerant yeasts in controlled simultaneous saccharifications and fermentations of cellulose to ethanol

Simultaneous saccharification and fermentation (SSF) experiments were performed at selected temperatures (37, 41, and 43 degrees C) to obtain comprehensive material balance and performance data for several promising strains of thermotolerant yeast. Parameters measured were ethanol concentration, yeast cell density, and residual sugar and cellulose concentrations. The three yeasts Saccharomyces uvarum, Candida brassicae, and C. lusitaniae and two mixed cultures of Brettanomyces clausenii with S. cerevisiae (mixed culture I) and C. Iusitaniae with S. uvarum (mixed culture II) exhibited rapid rates of fermentation, high ethanol yields, strong viability, or high cellobiase activity. Overall, mixed culture II at 41 degrees C performed better than either component yeast by themselves because it combined a cellobiose fermenting capability with the high ethanol tolerance and rapid glucose fermentation of conventional industrial yeasts. Thus, the mixed cultures provide good initial rates by preventing buildup of cellobiose (a strong inhibitor of enzyme activity) while attaining high ultimate yields of ethanol for high cellulase concentrations. However, C. brassicae and S. uvarum gave similar results to mixed culture II at 37 degrees C.     

Fermentation of molasses by Zymomonas mobilis: effects of temperature and sugar concentration on ethanol production

Fermentations utilizing strains of Zymomonas mobilis, in place of the traditional yeasts, have been proposed due their ethanol yields being close to theoretical. Ethanol production from sugar cane molasses was analyzed under different culture conditions using Z. mobilis in batch fermentation. The total reducing sugars (TRS) concentrations in the molasses, temperature, agitation and culture time effects were studied simultaneously through factorial design. The best conditions for ethanol production were 200 g L(-1) of total reducing sugars in the molasses, temperature of 30 degrees C and static culture and time of fermentation of 48 h, achieving 55.8 g L(-1). The pH of the medium was kept constant during the experiments, showing that molasses presents a buffering effect.     

Decolorization of alcohol distillery wastewater by thermotolerant white-rot fungi

To detect thermotolerant fungus strain for decolorization of alcohol distillery wastewater (WAD), 38 fungus strains were studied. Ability of ligninolytic enzyme production was examined at 35 and 43 degrees C on agar media containing 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) and MnCl2. At 43 degrees C, four of Pycnoporus coccineus strains showed their higher potential for WAD decolorization both on agar media and in liquid media. Immobilized mycelia on polyurethane foam removed total phenol about threefold higher than free mycelia did in shaking condition at 43 degrees C. Moreover, color removed by immobilized mycelia nearly 50% higher than free mycelia did.     

Production of ethanol from guava pulp by yeast strains

Guava pulp used for ethanol production by three yeast strains contained 10% (w/v) total sugars and was pH 4.1. Ethanol production at the optimum sugar concentration of 10%, at pH 4.1 and 30°C was 1.5%, 3.6% and 3.9% (w/v) by Saccharomyces cerevisiae MTCC 1972, Isolate-1 and Isolate-2, respectively, at 60 h fermentation. Higher sugar concentrations at 15 and 20% were inhibitory for ethanol production by all test cultures. The maximum production of ethanol at optimum natural sugar concentration (10%) of guava pulp, was 5.8% (w/v) at pH 5.0 by Isolate-2 over 36 h fermentation, which was only slightly more than the quantity of ethanol produced by Saccharomyces cerevisiae (5.0%) and Isolate-1 (5.3%) over 36 and 60h fermentation, respectively.     

Production of ethanol from molasses at 45 °C using alginate-immobilized Kluyveromyces marxianus imb3

The thermotolerant, ethanol-producing yeast strain, Kluyveromyces marxianus IMB3, has been immobilized in calcium alginate matrices. The ability of the biocatalyst to produce ethanol from cane molasses originating in Guatemala, Honduras, Senegal, Guyana and the Philippines was examined. In each case the molasses was diluted to yield a sugar concentration of 140?g/l and fermentations were carried out in batch-fed mode at 45?°C. During the first 24 hours, the maximum ethanol concentrations obtained ranged from 43–57?g/l with optimum production on the molasses from Honduras. Ethanol production during subsequent re-feeding of the fermentations at 24-hour intervals over a 120-hour period, decreased steadily to concentrations ranging from 20–36?g/l and it was found that ethanol productivity remained highest in fermentations containing the molasses from Guyana. When each set of fermentations was re-fed at 120?h and allowed to continue for 48?h, ethanol production again increased to a maximum with concentrations ranging from 25–52?g/l. It was also found however, that increasing the time between re-feeding at this stage in fermentation had a detrimental effect on the functionality of the biocatalyst.     

Cauliflower waste incorporation into cane molasses improves ethanol production using <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> MTCC 178

Diluted cane molasses having total sugar and reducing sugar content of 9.60 and 3.80% (w/v) respectively was subjected to ethanol production by Saccharomyces cerevisiae MTCC 178. Incorporation of dried Cauliflower Waste (CW) in molasses at the level of 15 % increased ethanol production by nearly 36 % compared to molasses alone. Addition of 0.2 % yeast extract improved ethanol production by nearly 49 % as compared to molasses alone. When the medium containing diluted molasses and 0.2 % yeast extract was supplemented with 15 % CW, 29 % more ethanol was produced compared to molasses with 0.2 % yeast extract. Cell biomass, ethanol production, final ethanol concentration and fermentation efficiency of 2.65 mg mL⁻¹, 41.2 gL⁻¹, 0.358 gg⁻¹ and 70.11 % respectively were found to be best at 15% CW supplementation level besides reduction in fermentation time but further increase in CW level resulted in decline on account of all the above parameters. This is probably the first report to our knowledge, in which CW was used in enhancing ethanol production significantly using a small quantity of yeast extract.     

Ethanol production at 45 °C by Kluyveromyces marxianus IMB3 during growth on molasses pre-treated with Amberlite® and non-living biomass

The use of high concentrations of molasses as a fermentation feed-stock for ethanol production is normally precluded by the presence of inhibitory compounds. Use of the thermotolerant, ethanol-producing yeast strain Kluyveromyces marxianus IMB3 in fermentations containing high concentrations of molasses resulted in sub-optimal production of ethanol. The results suggested that this was caused by the presence of inhibitory materials rather than an intolerance to increased concentrations of ethanol. In the current study we describe the pretreatment of molasses preparations with either an Amberlite^® monobed mixed ion-exchange resin or non-living microbial biomass from a local distillery. In the study molasses samples diluted to yield a final sugar concentration of 160?g/l were used as the substrate. Control fermentations using the untreated molasses dilutions yielded a maximum ethanol concentration of 40?g/l, representing 49% of the maximum theoretical yield. Fermentations using molasses samples pre-treated with Amberlite^® or non-living biomass yielded maximum ethanol concentrations of 58 and 54?g/l, representing 71 and 66% of the maximum theoretical yield, respectively. The results suggest that pre-treatment brings about removal of toxic or inhibitory materials from the fermentation feed-stock and we believe that such pre-treatments, particularly using the less expensive non-living biomass preparations may find a role in processes concerned with the commercial production of ethanol from molasses using this microorganism.     

Growth and fermentation characteristics of new selected strains of Saccharomyces at high temperatures and high cell densities

Maintenance of high cell viability was the main characteristic of our new strains of thermotolerant Saccharomyces. Total sugar conversion to ethanol was observed for sugarcane juice fermentation at 38-40 degrees C in less than 10 h and without continuous aeration of the culture. Invertase activity differed among the selected strains and increased during fermentation but was not dependent on cell viability. Invertase activity of the cells and optimum temperature for growth, as well as velocity of ethanol formation, were dependent on medium composition and the type of strain used. At high sugarcane syrup concentrations, the best temperature for ethanol formation by strain 781 was 35 degrees C. Distinct differences among the velocities of ethanol production using selected strains were also observed in sugarcane syrup at 35-38 degrees C.     

Isolation and improvement of a thermotolerant Saccharomyces cerevisiae strain

The ambient temperature is a drawback in industrial ethanol production in Jaffna due to heat killing of yeast during fermentation. Thus a search was initiated for thermotolerant organisms suitable for fermentation in hot climates. The screening of the best wild-type organisms was undertaken as the first step. Thermotolerant strains were selected from environments where there are chances of organisms being exposed to high temperature. The samples were enriched and screened for thermotolerant organisms which survived at 45 °C for 15 h. Among the yeast strains selected from different sources, thermotolerant strains with the capacity to withstand 45 °C for 15 h were found in samples collected from the compost heap and distillery environments. Three colonies from the distillery environment were selected for further studies and named p1, p2 and p3. Exponential phase (18 h) cultures of p1, p2 and p3 were subjected to 15 temperature treatment cycles (at 50 °C each for 3 h) and thermally adapted strains pt1, pt2 and pt3 were obtained, showing 100, 30 and 20% viability at 50 °C for 30 min respectively. The initial round of thermal adaptation cycles increased the duration of 100% viability from 20 h (p1) to 68 h (pt1) when incubated at 40 °C. Very little benefit was obtained when pt1 was treated with u.v. and ethyl methanesulphonate. The selected strain was identified and designated as Saccharomyces cerevisiae S1. The ethanol produced from 100 g glucose l^–1 by S. cerevisiae S1 was 46 g l^–1 (36 h), 38 g l^–1 (48 h) and 26 g l^–1 (48 h) at 40, 43 and 45 °C respectively in rich nutrient medium.     

Ethanol production from henequen (Agave fourcroydes Lem.) juice and molasses by a mixture of two yeasts

In the fermentation process of henequen (Agave fourcroydes Lem.) leaf juice, complemented with industrial molasses, the use of an inoculum comprising two yeasts: Kluyveromyces marxianus (isolated from the henequen plant) and Saccharomyces cerevisiae (commercial strain) was studied. An ethanol production of 5.22+/-1.087% v/v was obtained. Contrary to expected, a decrease on ethanol production was observed with the use of the K. marxianus strain. The best results were obtained when a mixture of 25% K. marxianus and 75% S. cerevisiae or S. cerevisiae alone were used with an initial inoculum concentration of 3x10(7)cellmL(-1). Furthermore, it was possible to detect a final concentration of approximately 2-4gL(-1) of reducing sugars that are not metabolized by the yeasts for the ethanol production. These results show that although the use of a mixture of yeasts can be of interest for the production of alcoholic beverages, it can be the opposite in the case of ethanol production for industrial purposes where manipulation of two strains can raise the production costs.     

Industrial scale ethanol production using the thermotolerant yeast Kluyveromyces marxianus IMB3 in an Indian distillery

The thermotolerant ethanol producing Kluyveromyces marxianus IMB3 yeast was used in eight 60m3 fermenters for industrial ethanol production in India using sugarcane molasses. Ethanol ranged between 6.0–7.2% (w/v) with added advantages of elimination of cooling during fermentation and shorter fermentation periods of 20h. © Rapid Science Ltd. 1998