The fermentation of sugarcane molasses by Dekkera bruxellensis and the mobilization of reserve carbohydrates

The yeast Dekkera bruxellensis is considered to be very well adapted to industrial environments, in Brazil, USA, Canada and European Countries, when different substrates are used in alcoholic fermentations. Our previous study described its fermentative profile with a sugarcane juice substrate. In this study, we have extended its physiological evaluation to fermentation situations by using sugarcane molasses as a substrate to replicate industrial working conditions. The results have confirmed the previous reports of the low capacity of D. bruxellensis cells to assimilate sucrose, which seems to be the main factor that can cause a bottleneck in its use as fermentative yeast. Furthermore, the cells of D. bruxellensis showed a tendency to deviate most of sugar available for biomass and organic acids (lactic and acetic) compared with Saccharomyces cerevisiae, when calculated on the basis of their respective yields. As well as this, the acetate production from molasses medium by both yeasts was in marked contrast with the previous data on sugarcane juice. Glycerol and ethanol production by D. bruxellensis cells achieved levels of 33 and 53 % of the S. cerevisiae, respectively. However, the ethanol yield was similar for both yeasts. It is worth noting that this yeast did not accumulate trehalose when the intracellular glycogen content was 30 % lower than in S. cerevisiae. The lack of trehalose did not affect yeast viability under fermentation conditions. Thus, the adaptive success of D. bruxellensis under industrial fermentation conditions seems to be unrelated to the production of these reserve carbohydrates.     

Production of <Emphasis Type="SmallCaps">d</Emphasis>-lactic acid from sugarcane molasses,sugarcane juice and sugar beet juice by <Emphasis Type="Italic">Lactobacillus delbrueckii</Emphasis>

Lactobacillus delbrueckii was grown on sugarcane molasses, sugarcane juice and sugar beet juice in batch fermentation at pH 6 and at 40°C. After 72 h, the lactic acid from 13% (w/v) sugarcane molasses (119 g total sugar l⁻¹) and sugarcane juice (133 g total sugar l⁻¹) was 107 g l⁻¹ and 120 g l⁻¹, respectively. With 10% (w/v) sugar beet juice (105 g total sugar l⁻¹), 84 g lactic acid l⁻¹ was produced. The optical purities of d-lactic acid from the feedstocks ranged from 97.2 to 98.3%.     

An innovative consecutive batch fermentation process for very high gravity ethanol fermentation with self-flocculating yeast

An innovative consecutive batch fermentation process was developed for very high gravity (VHG) ethanol fermentation with the self-flocculating yeast under high biomass concentration conditions. On the one hand, the high biomass concentration significantly shortened the time required to complete the VHG fermentation and the duration of yeast cells suffering from strong ethanol inhibition, preventing them from losing viability and making them suitable for being repeatedly used in the process. On the other hand, the separation of yeast cells from the fermentation broth by sedimentation instead of centrifugation, making the process economically more competitive. The VHG medium composed of 255 g L⁻¹ glucose and 6.75 g L⁻¹ each of yeast extract and peptone was fed into the fermentation system for nine consecutive batch fermentations, which were completed within 8–14 h with an average ethanol concentration of 15% (v/v) and ethanol yield of 0.464, 90.8% of its theoretical value of 0.511. The average ethanol productivity that was calculated with the inclusion of the downstream time for the yeast flocs to settle from the fermentation broth and the supernatant to be removed from the fermentation system was 8.2 g L⁻¹ h⁻¹, much higher than those previously reported for VHG ethanol fermentation and regular ethanol fermentation with ethanol concentration around 12% (v/v) as well.     

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.     

Use of sugarcane molasses “B” as an alternative for ethanol production with wild-type yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> ITV-01 at high sugar concentrations

Molasses “B” is a rich co-product of the sugarcane process. It is obtained from the second step of crystallization and is richer in fermentable sugars (50–65%) than the final molasses, with a lower non-sugar solid content (18–33%); this co-product also contains good vitamin and mineral levels. The use of molasses “B” for ethanol production could be a good option for the sugarcane industry when cane sugar prices diminish in the market. In a complex medium like molasses, osmotolerance is a desirable characteristic for ethanol producing strains. The aim of this work was to evaluate the use of molasses “B” for ethanol production using Saccharomyces cerevisiae ITV-01 (a wild-type yeast isolated from sugarcane molasses) using different initial sugar concentrations (70–291 g L⁻¹), two inoculum sizes and the addition of nutrients such as yeast extract, urea, and ammonium sulphate to the culture medium. The results obtained showed that the strain was able to grow at 291 g L⁻¹ total sugars in molasses “B” medium; the addition of nutrients to the culture medium did not produce a statistically significant difference. This yeast exhibits high osmotolerance in this medium, producing high ethanol yields (0.41 g g⁻¹). The best conditions for ethanol production were 220 g L⁻¹ initial total sugars in molasses “B” medium, pH 5.5, using an inoculum size of 6 × 10⁶ cell mL⁻¹; ethanol production was 85 g L⁻¹, productivity 3.8 g L⁻¹ h⁻¹ with 90% preserved cell viability.     

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.     

Fermentation of high concentrations of lactose to ethanol by engineered flocculent Saccharomyces cerevisiae

The development of microorganims that efficiently ferment lactose has a high biotechnological interest, particularly for cheese whey bioremediation processes with simultaneous bio-ethanol production. The lactose fermentation performance of a recombinant Saccharomyces cerevisiae flocculent strain was evaluated. The yeast consumed rapidly and completely lactose concentrations up to 150 g l⁻¹ in either well- or micro-aerated batch fermentations. The maximum ethanol titre was 8% (v/v) and the highest ethanol productivity was 1.5–2 g l⁻¹ h⁻¹, in micro-aerated fermentations. The results presented here emphasise that this strain is an interesting alternative for the production of ethanol from lactose-based feedstocks.     

Production of bacterial cellulose from <Emphasis Type="Italic">Enterobacter amnigenus</Emphasis> GH-1 isolated from rotten apple

Bacterial cellulose finds novel applications in biomedical, biosensor, food, textile and other industries. The optimum fermentation conditions for the production of cellulose by newly isolated Enterobacter amnigenus GH-1 were investigated. The strain was able to produce cellulose at temperature 25–35°C with a maximum at 28°C. Cellulose production occurred at pH 4.0–7.0 with a maximum at 6.5. After 14 days of incubation, the strain produced 2.5 g cellulose/l in standard medium whereas cellulose yield in the improved medium was found to be 4.1 g/l. The improved medium consisted of 4% (w/v) fructose, 0.6% (w/v) casein hydrolysate, 0.5% (w/v) yeast extract, 0.4% (w/v) disodium phosphate, and 0.115% (w/v) citrate. Addition of metal ions like zinc, magnesium, and calcium and solvents like methanol and ethanol were found to be stimulatory for cellulose production by the strain. The strain used natural carbon sources like molasses, starch hydrolysate, sugar cane juice, coconut water, coconut milk, pineapple juice, orange juice, and pomegranate juice for growth and cellulose production. Fruit juices can play important role in commercial exploitation of bacterial cellulose by lowering the cost of the production medium.     

Ethanol production from corn cob hydrolysates by <Emphasis Type="Italic">Escherichia coli</Emphasis> KO11

Corn cob hydrolysates, with xylose as the dominant sugar, were fermented to ethanol by recombinant Escherichia coli KO11. When inoculum was grown on LB medium containing glucose, fermentation of the hydrolysate was completed in 163 h and ethanol yield was 0.50 g ethanol/g sugar. When inoculum was grown on xylose, ethanol yield dropped, but fermentation was faster (113 h). Hydrolysate containing 72.0 g/l xylose and supplemented with 20.0 g/l rice bran was readily fermented, producing 36.0 g/l ethanol within 70 h. Maximum ethanol concentrations were not higher for fermentations using higher cellular concentration inocula. A simulation of an industrial process integrating pentose fermentation by E. coli and hexose fermentation by yeast was carried out. At the first step, E. coli fermented the hydrolysate containing 85.0 g/l xylose, producing 40.0 g/l ethanol in 94 h. Baker's yeast and sucrose (150.0 g/l) were then added to the spent fermentation broth. After 8 h of yeast fermentation, the ethanol concentration reached 104.0 g/l. This two-stage fermentation can render the bioconversion of lignocellulose to ethanol more attractive due to increased final alcohol concentration. Journal of Industrial Microbiology & Biotechnology (2002) 29, 124–128 doi:10.1038/sj.jim.7000287 Received 20 February 2002/ Accepted in revised form 04 June 2002     

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 microbial levan from sucrose,sugarcane juice and beet molasses

Summary Bacillus polymyxa (NRRL-18475) produced a levan-type fructan (B, 26 fructofuranoside) when grown on sucrose, sugarcane juice, and sugarbeet molasses. The organism converted about 46% of the fructose moiety of sucrose to levan when grown on sucrose medium, however, the yields of levan from sugarcane juice and beet molasses were much less than sucrose solution. Such sugarcane juice and beet molasses can be made a good substrate for levan production by various modifications. Adding peptone to sugarcane juice or passing beet molasses through a column of gel filtration media improved levan yield to a level almost comparable to that obtained from sucrose.     

Optimization of temperature,sugar concentration,and inoculum size to maximize ethanol production without significant decrease in yeast cell viability

Aiming to obtain rapid fermentations with high ethanol yields and a retention of high final viabilities (responses), a 2³ full-factorial central composite design combined with response surface methodology was employed using inoculum size, sucrose concentration, and temperature as independent variables. From this statistical treatment, two well-fitted regression equations having coefficients significant at the 5% level were obtained to predict the viability and ethanol production responses. Three-dimensional response surfaces showed that increasing temperatures had greater negative effects on viability than on ethanol production. Increasing sucrose concentrations improved both ethanol production and viability. The interactions between the inoculum size and the sucrose concentrations had no significant effect on viability. Thus, the lowering of the process temperature is recommended in order to minimize cell mortality and maintain high levels of ethanol production when the temperature is on the increase in the industrial reactor. Optimized conditions (200 g/l initial sucrose, 40 g/l of dry cell mass, 30 °C) were experimentally confirmed and the optimal responses are 80.8 ± 2.0 g/l of maximal ethanol plus a viability retention of 99.0 ± 3.0% for a 4-h fermentation period. During consecutive fermentations with cell reuse, the yeast cell viability has to be kept at a high level in order to prevent the collapse of the process.     

Interaction of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>–<Emphasis Type="Italic">Lactobacillus fermentum</Emphasis>–<Emphasis Type="Italic">Dekkera bruxellensis</Emphasis> and feedstock on fuel ethanol fermentation

The alcoholic fermentation for fuel ethanol production in Brazil occurs in the presence of several microorganisms present with the starter strain of Saccharomyces cerevisiae in sugarcane musts. It is expected that a multitude of microbial interactions may exist and impact on the fermentation yield. The yeast Dekkera bruxellensis and the bacterium Lactobacillus fermentum are important and frequent contaminants of industrial processes, although reports on the effects of both microorganisms simultaneously in ethanolic fermentation are scarce. The aim of this work was to determine the effects and interactions of both contaminants on the ethanolic fermentation carried out by the industrial yeast S. cerevisiae PE-2 in two different feedstocks (sugarcane juice and molasses) by running multiple batch fermentations with the starter yeast in pure or co-cultures with D. bruxellensis and/or L. fermentum. The fermentations contaminated with D. bruxellensis or L. fermentum or both together resulted in a lower average yield of ethanol, but it was higher in molasses than that of sugarcane juice. The decrease in the CFU number of S. cerevisiae was verified only in co-cultures with both D. bruxellensis and L. fermentum concomitant with higher residual sucrose concentration, lower glycerol and organic acid production in spite of a high reduction in the medium pH in both feedstocks. The growth of D. bruxellensis was stimulated in the presence of L. fermentum resulting in a more pronounced effect on the fermentation parameters than the effects of contamination by each microorganism individually.     

Influence of cultivation procedure for <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> used as pitching agent in industrial spent sulphite liquor fermentations

The cell viability and fermentation performance often deteriorate in fermentations of spent sulphite liquor (SSL). This investigation therefore addresses the question of how different cultivation conditions for yeast cells influence their ability to survive and boost the ethanol production capacity in an SSL-based fermentation process. The strains used as pitching agents were an industrially harvested Saccharomyces cerevisiae and commercial dry baker’s yeast. This study therefore suggests that exposure to SSL in combination with nutrients, prior to the fermentation step, is crucial for the performance of the yeast. Supplying 0.5 g/l fresh yeast cultivated under appropriate cultivation conditions may increase ethanol concentration more than 200%.     

Chromium uptake bySaccharomyces cerevisiae and isolation of glucose tolerance factor from yeast biomass

Fermentations with yeastSaccharomyces cerevisiae in semiaerobic and in static conditions with the addition of chromic chloride into the used molasses medium were analysed. It was proved that the addition of optimal amounts of CrCl₃ into the basal medium enhanced the kinetics of alcohol fermentations. The addition of 200 mg/l CrCl₃ into the medium stimulated both the yeast growth and the ethanol production in all experimental conditions. On the other hand, the results showed that Cr³⁺ ions were incorporated into yeast cells during fermentation. Under these conditions the accumulation of Cr³⁺ ions was performed by yeast cells during the exponential growth phase, and with enriched amounts of 30–45 (μg/g_d.m. of cells. Yeast biomass enriched with chromium ions was extracted with 01 mol/l NH₄OH assuming that the extracts had the glucose tolerance factor (GTF). Then the extracts were passed through a gel-filtration column in order to isolate and purify the GTF. The presence of GTF in the purified fractions was determined by measuring the absorbance at 260 nm. It is evident from the obtained results that the added purified fractions enhanced the rates of CO₂ production as well as the glucose utilization during alcoholic fermentation. As expected, the enhancement of both rates depended on the amounts of extracts added to the fermentation substrate. Thus, it is evident that purified extracts contained the GTF compound, and that Cr³⁺ ions were bonded to the protein molecule.     

Novel strategy using an adsorbent-column chromatography for effective ethanol production from sugarcane or sugar beet molasses

Molasses-based distilleries generate large volumes of a highly polluted and dark brown-colored wastewater. The present work describes the way in which an adsorbent-column chromatography can effectively remove the colorant and produce biomass ethanol from sugarcane or sugar beet molasses. It was found that the color and chemical oxygen demand of the resulting wastewater was respectively reduced by approximately 87% and 28% as compared with conventional molasses fermentation. Gas chromatography showed that the decolorized molasses maintained good ethanol productivity almost equal to that of the original molasses. Furthermore, it was revealed that the colorant concentrations of about 5 mg ml⁻¹ in the medium were the most favorable for ethanolic fermentation. In summary, we have concluded that this method is the most effective when the adsorbent chromatography is performed just before molasses fermentation and that the decolorized molasses is an ideal substrate for fuel ethanol production.     

Feasibility of ethanol production from coffee husks

The objective of this work was to evaluate the feasibility of ethanol production by fermentation of coffee husks by Saccharomyces cerevisiae. Batch fermentation studies were performed employing whole and ground coffee husks, and aqueous extract from ground coffee husks. It was observed that fermentation yield decreased with an increase in yeast concentration. The best results were obtained for the following conditions: whole coffee husks, 3 g yeast/l substrate, temperature of 30°C. Under these conditions ethanol production was 8.49 ± 0.29 g/100 g dry basis (13.6 ± 0.5 g ethanol/l), a satisfactory value in comparison to literature data for other residues such as corn stalks, barley straw and hydrolyzed wheat stillage (5–11 g ethanol/l). Such results indicate that coffee husks present excellent potential for residue-based ethanol production.     

Anaerobic thermophilic fermentation for carboxylic acid production from in-storage air-lime-treated sugarcane bagasse

Wet storage and in situ lime pretreatment (50 °C, 1-atm air, 56 days, excess lime loading of 0.3 g Ca(OH)₂/g dry biomass) of sugarcane bagasse (4,000 g dry weight) was performed in a bench-scale pile pretreatment system. Under thermophilic conditions (55 °C, NH₄HCO₃ buffer, methane inhibitors), air-lime-treated bagasse (80 wt.%) and chicken manure (20 wt.%) were anaerobically co-digested in 1-L rotary fermentors by a mixed culture of marine microorganisms (Galveston, TX). During four-stage countercurrent fermentation, the resulting carboxylic acids consisted of primarily acetate (average 87.7 wt.%) and butyrate (average 9.0 wt.%). The experimental fermentation trains had the highest yield (0.47 g total acids/g volatile solids (VS) fed) and highest selectivity (0.79 g total acids/g VS digested) at a total acid concentration of 28.3 g/L, which is equivalent to an ethanol yield of 105.2 gal/(tonne VS fed). Both high total acid concentrations (>44.7 g/L) and high substrate conversions (>77.5%) are predicted for countercurrent fermentations of bagasse at commercial scale, allowing for an efficient conversion of air-lime-treated biomass to liquid transportation fuels and chemicals via the carboxylate platform.     

Production of bioethanol from organic whey using <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis>

Ethanol production by K. marxianus in whey from organic cheese production was examined in batch and continuous mode. The results showed that no pasteurization or freezing of the whey was necessary and that K. marxianus was able to compete with the lactic acid bacteria added during cheese production. The results also showed that, even though some lactic acid fermentation had taken place prior to ethanol fermentation, K. marxianus was able to take over and produce ethanol from the remaining lactose, since a significant amount of lactic acid was not produced (1–2 g/l). Batch fermentations showed high ethanol yield (~0.50 g ethanol/g lactose) at both 30°C and 40°C using low pH (4.5) or no pH control. Continuous fermentation of nonsterilized whey was performed using Ca-alginate-immobilized K. marxianus. High ethanol productivity (2.5–4.5 g/l/h) was achieved at dilution rate of 0.2/h, and it was concluded that K. marxianus is very suitable for industrial ethanol production from whey.     

beta-Carotene production in sugarcane molasses by a Rhodotorula glutinis mutant

Several wild strains and mutants of Rhodotorula spp. were screened for growth, carotenoid production and the proportion of -carotene produced in sugarcane molasses. A better producer, Rhodotorula glutinis mutant 32, was optimized for carotenoid production with respect to total reducing sugar (TRS) concentration and pH. In shake flasks, when molasses was used as the sole nutrient medium with 40 g l⁻¹ TRS, at pH 6, the carotenoid yield was 14 mg l⁻¹ and -carotene accounted for 70% of the total carotenoids. In a 14-l stirred tank fermenter, a 20% increase in torulene content was observed in plain molasses medium. However, by addition of yeast extract, this effect was reversed and a 31% increase in -carotene content was observed. Dissolved oxygen (DO) stat fed-batch cultivation of mutant 32 in plain molasses medium yielded 71 and 185 mg l⁻¹ total carotenoids in double- and triple-strength medium, respectively. When supplemented with yeast extract, the yields were 97 and 183 mg l⁻¹ total carotenoid with a 30% increase in -carotene and a simultaneous 40% decrease in torulene proportion. Higher cell mass was also achieved by double- and triple-strength fed-batch fermentation. Journal of Industrial Microbiology & Biotechnology (2001) 26, 327–332. Received 18 September 2000/ Accepted in revised form 02 March 2001