Isolation of thermotolerant ethanologenic yeasts and use of selected strains in industrial scale fermentation in an Egyptian distillery

An enrichment and isolation program for new ethanol-producing thermotolerant yeasts as well as a screening program of some known thermotolerant strains resulted in the selection of several strains capable of growth at 40-43 degrees C. Among these strains four grew and fermented sugar cane molasses at 43 degrees C under batch conditions with sugar-conversion efficiencies >94% and ethanol concentrations 6.8-8.0% (w/v). The two best-performing strains, a Saccharomyces cerevisiae F111 and a Kluyveromyces marxianus WR12 were used in eight 87.5 m(3) fermentation runs (four using each strain) for industrial ethanol production in an Egyptian distillery using sugar cane molasses. Mean ethanol production was 7.7% and 7.4% (w/v), respectively, with an added advantage of cooling elimination during fermentation and higher ethanol yields compared to the distillery's S. cerevisiae SIIC (ATCC 24855) strain in use. The isolate S. cerevisiae F111 was subsequently adopted by the distillery for regular production with significant economical gains and water conservation.     

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.     

Continuous ethanol production by immobilized yeast reactor

Summary Saccharomyces cerevisiae yeast immobilized in calcium alginate gel beads was employed in packed-bed column reactors for continuous ethanol production from glucose or cane molasses, and for beer fermentation from barley malt wort. With properly balanced nutrient content or periodical regeneration of cells by nutrient addition and aeration, ethanol production could be maintained for several months. About 7 percent (w/v) ethanol content could be easily maintained with cane molasses diluted to about 17.5 percent (w/v) of total reducing sugars at about 4 to 5 h residence time. Beer of up to 4.5 percent (wv) of ethanol could be produced from barley wort at about 2 h residence time without any addition of nutrients.     

Pilot-scale multi-stage multi-feeding continuous ethanol fermentation using non-sterile cane molasses

Summary Non-aseptic fermentation of a 28 brix cane molasses solution was successfully carried out in a pilot-scale 5-stage multi-feeding continuous system for 30 days. The effluent ethanol concentration, overall volumetric productivity and sugar conversion yield averaged 8.54 % (v/v), 5.35 g/L-hr and 92.4 % of theoretical, respectively.     

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.     

Continuous Ethanol Fermentation of Pretreated Lignocellulosic Biomasses,Waste Biomasses,Molasses and Syrup Using the Anaerobic,Thermophilic Bacterium Thermoanaerobacter italicus Pentocrobe 411

Lignocellosic ethanol production is now at a stage where commercial or semi-commercial plants are coming online and, provided cost effective production can be achieved, lignocellulosic ethanol will become an important part of the world bio economy. However, challenges are still to be overcome throughout the process and particularly for the fermentation of the complex sugar mixtures resulting from the hydrolysis of hemicellulose. Here we describe the continuous fermentation of glucose, xylose and arabinose from non-detoxified pretreated wheat straw, birch, corn cob, sugar cane bagasse, cardboard, mixed bio waste, oil palm empty fruit bunch and frond, sugar cane syrup and sugar cane molasses using the anaerobic, thermophilic bacterium Thermoanaerobacter Pentocrobe 411. All fermentations resulted in close to maximum theoretical ethanol yields of 0.47–0.49 g/g (based on glucose, xylose, and arabinose), volumetric ethanol productivities of 1.2–2.7 g/L/h and a total sugar conversion of 90–99% including glucose, xylose and arabinose. The results solidify the potential of Thermoanaerobacter strains as candidates for lignocellulose bioconversion.     

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     

Lactobacillus reuteri CRL 1101 highly produces mannitol from sugarcane molasses as carbon source

Mannitol is a natural polyol extensively used in the food industry as low-calorie sugar being applicable for diabetic food products. We aimed to evaluate mannitol production by Lactobacillus reuteri CRL 1101 using sugarcane molasses as low-cost energy source. Mannitol formation was studied in free-pH batch cultures using 3-10% (w/v) molasses concentrations at 37?°C and 30?°C under static and agitated conditions during 48?h. L. reuteri CRL 1101 grew well in all assayed media and heterofermentatively converted glucose into lactic and acetic acids and ethanol. Fructose was used as an alternative electron acceptor and reduced it to mannitol in all media assayed. Maximum mannitol concentrations of 177.7?±?26.6 and 184.5?±?22.5?mM were found using 7.5% and 10% molasses, respectively, at 37?°C after 24-h incubation. Increasing the molasses concentration from 7.5% up to 10% (w/v) and the fermentation period up to 48?h did not significantly improve mannitol production. In agitated cultures, high mannitol values (144.8?±?39.7?mM) were attained at 8?h of fermentation as compared to static ones (5.6?±?2.9?mM), the highest mannitol concentration value (211.3?±?15.5?mM) being found after 24?h. Mannitol 2-dehydrogenase (MDH) activity was measured during growth in all fermentations assayed; the highest MDH values were obtained during the log growth phase, and no correlation between MDH activities and mannitol production was observed in the fermentations performed. L. reuteri CRL 1101 successfully produced mannitol from sugarcane molasses being a promising candidate for microbial mannitol synthesis using low-cost substrate.     

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.     

Production of Ethanol from Maltose by Zymobacter palmae Fermentation

The production of ethanol from maltose by Zymobacter palmae T109 in monoculture fermentations, and in co-culture fermentations together with Zymomonas mobilis B69 was studies. Zymobacter palmae T109, produced 5.5% (w/v) of ethanol when co-cultured with Zymomonas mobilis B69, but Zymobacter palmae T109 produced only 4.9% (w/v) ethanol from 15% (w/v) maltose medium in monoculture fermentation.     

Continuous ethanol fermentation using immobilized yeast cells

Growing cells of Saccharomyces cerevisiae immobilized in calcium alginate gel beads were employed in fluidizedbed reactors for continuous ethanol fermentation from cane molasses and other sugar sources. Some improvements were made in order to avoid microbial contamination and keep cell viability for stable long run operations. Notably, entrapment of sterol and unsaturated fatty acid into immobilized gel beads enhanced ethanol productivity more than 50 g ethanol/L gel h and prolonged life stability for more than one-half year. Cell concentration in the carrier was estimated over 250 g dry cell/L gel. A pilot plant with a total column volume of 4 kL was constructed and has been operated since 1982. As a result, it was confirmed that 8-10%(v/v)ethanol-containing broth was continuously produced from nonsterilized diluted cane molasses for over one-half year. The productivity of ethanol was calculated as 0.6 kL ethanol/kL reactor volume day with a 95% conversion yield versus the maximum theoretical yield for the case of 8.5% (v/v) ethanol broth.     

Sugarcane molasses and yeast powder used in the Fructooligosaccharides production by Aspergillus japonicus-FCL 119T and Aspergillus niger ATCC 20611

Different concentrations of sucrose (3–25% w/v) and peptone (2–5% w/v) were studied in the formulation of media during the cultivation of Aspergillus japonicus-FCL 119T and Aspergillus niger ATCC 20611. Moreover, cane molasses (3.5–17.5% w/v total sugar) and yeast powder (1.5–5% w/v) were used as alternative nutrients for both strains’ cultivation. These media were formulated for analysis of cellular growth, β-Fructosyltransferase and Fructooligosaccharides (FOS) production. Transfructosylating activity (U _t ) and FOS production were analyzed by HPLC. The highest enzyme production by both the strains was 3% (w/v) sucrose and 3% (w/v) peptone, or 3.5% (w/v) total sugars present in cane molasses and 1.5% (w/v) yeast powder. Cane molasses and yeast powder were as good as sucrose and peptone in the enzyme and FOS (around 60% w/w) production by studied strains.     

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.     

Conversion of corn fiber to ethanol by recombinant E. coli strain FBR3

Escherichia coli strain FBR3 that is an efficient biocatalyst for converting mixed sugar streams (eg, arabinose, glucose, and xylose) into ethanol. In this report, the strain was tested for conversion of corn fiber hydrolysates into ethanol. Corn fiber hydrolysates with total sugar concentrations of 7.5% (w/v) were prepared by reacting corn fiber with dilute sulfuric acid at 145°C. Initial fermentations of the hydrolysate by strain FBR3 had lag times of approximately 30 h judged by ethanol production. Further experiments indicated that the acetate present in the hydrolysate could not solely account for the long lag. The lag phase was greatly reduced by growing the pre-seed and seed cultures on corn fiber hydrolysate. Ethanol yields for the optimized fermentations were 90% of theoretical. Maximum ethanol concentrations were 2.80% w/v, and the fermentations were completed in approximately 50 h. The optimal pH for the fermentation was 6.5. Below this pH, sugar consumption was incomplete and above it, excess base addition was required throughout the fermentation. Two alternative neutralization methods (overliming and overliming with sulfite addition) have been reported for improving the fermentability of lignocellulosic hydrolysates. These methods further reduced the lag phase of the fermentation, albeit by a minor amount. Received 29 September 1998/ Accepted in revised form 20 February 1999     

Fermentation of molasses using a thermotolerant yeast,Kluyveromyces marxianus IMB3: simplex optimisation of media supplements

 The use of molasses as a substrate for ethanol production by the thermotolerant yeast Kluyveromyces marxianus var. marxianus was investigated at 45°C. A maximum ethanol concentration of 7.4% (v/v) was produced from unsupplemented molasses at a concentration of 23% (v/v). The effect on ethanol production of increasing the sucrose concentration in 23% (v/v) molasses was determined. Increased sucrose concentration had a similar detrimental effect on the final ethanol produced as the increase in molasses concentration. This indicated that the effect may be due to increased osmotic activity as opposed to other components in the molasses. The optimum concentration of the supplements nitrogen, magnesium, potassium and fatty acid for maximum ethanol production rate was determined using the Nelder and Mead (Computer J 7:308–313, 1965) simplex optimisation method. The optimum concentrations of the supplements were 0.576 g l^-1 magnesium sulphate, 0.288 g l^-1 potassium dihydrogen phosphate and 0.36% (v/v) linseed oil. Added nitrogen in the form of ammonium sulphate did not affect the ethanol production rate. Received: 29 January 1996/Received revision: 23 April 1996/Accepted: 29 April 1996     

Kissiris: A mineral support for the promotion of ethanol fermentation by Saccharomyces cerevisiae

Ethanol production using Saccharomyces cerevisiae, promoted by the mineral kissiris, is reported on. A three-fold increase of ethanol productivity in the fermentation of molasses was achieved. An ethanol yield factor 0.43 g/g and conversion of 93.3% at an initial sugar concentration (ISC) 208.5 g/l were obtained in the presence of this mineral in molasses fermentation, compared to 0.21 g/g and 44.2% in its absence. It is also shown that the fermentation of molasses takes place even at relatively higher ethanol levels, with kissiris contributing to a 35% reduction of the energy demand in grade-fuel and potable ethanol production. The proposed mineral was shown to have a smaller effect in fermentations carried out in synthetic media containing glucose or saccharose.     

Ethanol production from sugar cane segments in a high solids drum fermentor

Summary A successful yeast fermentation for the production of relatively high concentration of ethanol (9% w/v) was carried out using sugar cane segments. Extraction of sugar from segments occurred simultaneously with ethanol formation. The beer produced was transferred to a fresh batch of sugar cane segments and the fermentation cycle was repeated successively three times with the same beer. A high cane to water ratio was obtained in a rotating drum fermentor which allowed for a minimal amount of liquid to be used during the fermentation process.     

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.     

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.     

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.