Bacterial cellulose production by fed-batch fermentation in molasses medium

Batch and fed-batch fermentations for bacterial cellulose (BC) production using molasses as a carbon source by Acetobacter xylinum BPR2001 were carried out in a jar fermentor. For improvement of BC production, molasses was subjected to H2SO4-heat treatment. The maximum BC concentration by this treated molasses increased 76%, and the specific growth rate increased 2-fold compared with that by untreated molasses. In batch fermentation, when the initial sugar concentrations of H2SO4-heat-treated molasses were varied from 20 to 70 g/L, the highest value of maximum BC concentration of 5.3 g/L was observed at 20 g/L. BC production in intermittent fed-batch (IFB) fermentation was conducted referring to the data in batch fermentation, and the highest BC production of 7.82 g/L was obtained when 0.2 L of molasses medium was added five times. When continuous fed-batch (CFB) fermentations were conducted, maximum BC concentration was obtained with a feeding rate of 6.3 g-sugar/h, which was derived from the optimal IFB experiment.     

Determination of the best nutrient medium for the production of L-lactic acid from beet molasses a statistical approach

Optimization studies have been carried out for the production of L-lactic acid from the fermentation of beet molasses by Lactobacillus delbrueckii. A PLACKETT -BURMAN Design and a Central Composite Design have been used to determine the most suitable nutrient medium for obtaining a maximum cell concentration. A second-order polynomial empirical model relating both the cell and nutrient concentrations was formulated. The variables selected for the study were Yeast Extract, Peptone, Tween 80 (antifoam), MgSO₄ · 7H₂O, MnSO₄·4H₂O, FeSO₄ · 7H₂O and K₂HPO₄/KH₂PO₄. Among them, only Yeast Extract and Peptone were found to significantly affect the cell concentration. A maximum cell yield was found when the concentrations of Yeast Extract and Peptone were, respectively, 5.31 g/l and 5.08 g/l. All conclusions are restricted to the experimental range studied.     

Production of ethanol from molasses at 45 °C using Kluyveromyces marxianus IMB3 immobilized in calcium alginate gels and poly(vinyl alcohol) cryogel

The thermotolerant, ethanol-producing yeast strain Kluyveromyces marxianus IMB3 has been immobilized in calcium alginate gel and poly(vinyl alcohol) cryogel (PVAC) beads. The immobilized preparations were used as biocatalyst in fed-batch reactor systems for prolonged periods. The substrate utilized in each case consisted of sugar cane molasses diluted to yield a sugar load of 140?g/l. During the first cycle the maximum ethanol concentration produced by the alginate system was 57?g/l, representing 80% of the maximum theoretical yield. In the system employing the PVAC-immobilized biocatalyst, ethanol production increased to a maximum of 52–53?g/l, representing 73% of the maximum theoretical yield. In both cases, maximum ethanol concentration was achieved within a 72-hour period. When each system was operated on a fed-batch basis for a prolonged period of time the average ethanol concentrations produced in the alginate- and the PVAC-immobilized systems were 21 and 45?g/l, respectively. The results suggest that the PVAC-based immobilization system may provide a more practical alternative to alginate for the production of ethanol by K. marxianus IMB3 in continuous or semi-continuous fermentation systems.     

Microbial oil production from corncob acid hydrolysate by <Emphasis Type="Italic">Trichosporon cutaneum</Emphasis>

Corncob was treated by dilute H₂SO₄. The hydrolysate contained 45.7 g sugar/l. Without concentration or adding other nutrients, the hydrolysate, after being detoxified by overliming and adsorption with activated charcoal, was used for oil production using Trichosporon cutaneum. After 8 days’ growth in shake-flasks, the biomass was 22.1 g/l with a lipid content of 36%. The lipid yield per mass of sugar was 17.4% (w/w). Corncob thus is a promising raw material for microbial oil production by this yeast.     

Effect of acetic acid on astaxanthin production by Phaffia rhodozyma

Summary Low concentrations of acetic acid decreased the growth rate of and astaxanthin production by Phaffia rhodozyma on glucose, with growth completely inhibited by 2 g acetic acid/l. Using H₂SO₄ for pH control after sugar depletion caused a decline in the biomass concentration, whereas using acetic acid as titrant resulted in an increase in the biomass with a high astaxanthin content of 1430 g/g cells. An extended culture with a continuous glucose feed failed to maintain a high astaxanthin content.     

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.     

Utilization of Molasses Sugar for Lactic Acid Production by Lactobacillus delbrueckii subsp. delbrueckii Mutant Uc-3 in Batch Fermentation

Efficient lactic acid production from cane sugar molasses by Lactobacillus delbrueckii mutant Uc-3 in batch fermentation process is demonstrated. Lactic acid fermentation using molasses was not significantly affected by yeast extract concentrations. The final lactic acid concentration increased with increases of molasses sugar concentrations up to 190 g/liter. The maximum lactic acid concentration of 166 g/liter was obtained at a molasses sugar concentration of 190 g/liter with a productivity of 4.15 g/liter/h. Such a high concentration of lactic acid with high productivity from molasses has not been reported previously, and hence mutant Uc-3 could be a potential candidate for economical production of lactic acid from molasses at a commercial scale.     

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.     

Continuous ethanol production from molasses at 45 °C using alginate-immobilized Kluyveromyces marxianus IMB3 in a continuous-flow bioreactor

The thermotolerant, ethanol-producing yeast strain Kluyveromyces marxianus IMB3 was immobilized in calcium alginate and used in a continuous flow bioreactor to produce ethanol from molasses at 45?°C. The molasses was diluted to yield a number of final sugar concentrations and the effect of molasses sugar concentration on ethanol production by the continuous system was examined. Although maximum ethanol concentrations were obtained using sugar concentrations of 140?g/l, within 10?h of introducing the feed to the column bioreactors, those ethanol concentrations subsequently decreased to lower levels over a 48?h period. Examination of viable yeast cell number within the immobilization matrix indicated a dramatic reduction over this time period. At lower molasses concentrations, ethanol production by the continuous flow system remained relatively constant over this time period. In addition, the effect of residence time on ethanol production by the continuous flow bioreactor was examined at a fixed molasses sugar concentration (120?g/l) and a residence time of 0.66?h was found to be optimal on the basis of volumetric productivity. Efficiencies of the continuous flow bioreactor configuration used in these studies ranged from 31–76%.     

Production of Fructose and Ethanol from Cane Molasses Using Saccharomyces cerevisiae ATCC 36858

The purpose of this research was to study the possibility of the production of ethanol and enriched fructose syrups from sugar cane molasses using the yeast Saccharomyces cerevisiae ATCC 36858. In batch experiments with a total sugar concentration of between 96.7 g/l and 323.5 g/l, the fructose yield was above 90% of the theoretical value. The ethanol yield and volumetric productivity were in the range of 66% and 77% of the theoretical value, and between 0.53 g ethanol/l × h and 3.15 g ethanol/l × h, respectively. The fructose fraction in the carbohydrates content of the produced syrups was more than 95% when the total initial sugar concentration in the medium was below 273.8 g/l. Some oligosaccharides and glycerol were also produced in all tested media. The maximum amount of produced oligosaccharides including raffinose accounted for 13.4 g/l in the cane molasses medium with 323.5 g/l sugars in the initial phase of the fermentation process. The oligosaccharides produced and raffinose were completely consumed by the end of the fermentation process when the total initial sugar concentration was less than 191.3 g/l. The glycerol concentration was below 9.9 g/l. These findings are useful in the production of ethanol and high fructose syrups using sugar cane molasses.     

Optimization of lactic acid production from beet molasses by Lactobacillus delbrueckii NCIMB 8130

Production of lactic acid from beet molasses by Lactobacillus delbrueckii NCIMB 8130 in static and shake flask fermentation was investigated. Shake flasks proved to be a better fermentation system for this purpose. Substitution of yeast extract with other low cost protein sources did not improve lactic acid production. The maximum lactic acid concentration was achieved without treatment of molasses. A Central Composite Design was employed to determine the maximum lactic acid concentration at optimum values for the process variables (sucrose, yeast extract, CaCO₃). A satisfactory fit of the model was realized. Lactic acid production was significantly affected both by sucrose–yeast extract and sucrose–CaCO₃ interactions, as well as by the negative quadratic effects of these variables. Sucrose and yeast extract had a linear effect on lactic acid production while the CaCO₃ had no significant linear effect. The maximum lactic acid concentration (88.0 g/l) was obtained at concentrations for sucrose, yeast extract and CaCO₃ of 89.93, 45.71 and 59.95 g/l, respectively.     

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     

Effect of pretreatment chemicals on xylose fermentation by Pichia stipitis

Pretreatment of biomass with dilute H₂SO₄ results in residual acid which is neutralized with alkalis such as Ca(OH)₂, NaOH and NH₄OH. The salt produced after neutralization has an effect on the fermentation of Pichia stipitis. Synthetic media of xylose (60 g total sugar/l) was fermented to ethanol in the presence and absence of the salts using P. stipitis CBS 6054. CaSO₄ enhanced growth and xylitol production, but produced the lowest ethanol concentration and yield after 140 h. Na₂SO₄ inhibited xylitol production, slightly enhanced growth towards the end of fermentation but had no significant effect on xylose consumption and ethanol concentration. (NH₄)₂SO₄ inhibited growth, had no effect on xylitol production, and enhanced xylose consumption and ethanol production.     

Citric acid production from sugar cane molasses by 2-deoxyglucose-resistant mutant strain ofAspergillus niger

Citric acid production from sugar cane molasses byAspergillus niger NIAB 280 was studied in a batch cultivation process. A maximum of 90 g/L total sugar was utilized in citric acid production medium. From the parental strainA. niger, mutant strains showing resistance to 2-deoxyglucose in Vogal's medium containing molasses as a carbon source were induced by γ-irradiation. Among the new series of mutant strains, strain RP7 produced 120 g/L while the parental strain produced 80 g/L citric acid (1.5-fold improvement) from 150 g/L of molasses sugars. The period of citric acid production was shortened from 10 d for the wild-type strain to 6–7 d for the mutant strain. The efficiency of substrate uptake rate with respect to total volume substrate consumption rate,Q _s (g per L per h) and specific substrate consumption rate,q _s (g substrate per g cells per h) revealed that the mutant grew faster than its parent. This indicated that the selected mutant is insensitive to catabolite repression by higher concentrations of sugars for citric acid production. With respect to the product yield coefficient (Y _p/x), volume productivity (Q _p) and specific product yields (q _p), the mutant strain is significantly (p≤0.05) improved over the parental strain.     

Mutation of Aspergillus niger for hyperproduction of citric acid from black strap molasses

Spore suspensions of Aspergillus niger GCB 75, which produced 31.1 g/l citric acid from 15% sugars in molasses, were subjected to u.v.-induced mutagenesis. Among three variants, GCM 45 was found to be the best citric acid producer and was further improved by chemical mutagenesis using NTG. Out of 3 deoxy-D-glucose-resistant variants, GCM 7 was selected as the best mutant which produced 86.1 ± 1.5 g/l citric acid after 168 h of fermentation of potassium ferricyanide + H₂SO₄-pretreated black strap molasses (containing 150 g sugars/l) in Vogel's medium. On the basis of comparison of kinetic parameters, namely the volumetric substrate uptake rate (Q _s), and specific substrate uptake rate (q _s), the volumetric productivity, theoretical yield and specific product formation rate, it was observed that the mutants were faster growing organisms and had the ability to overproduce citric acid.     

Multicomponent cellulase production by Cellulomonas biazotea NCIM‐2550 and its applications for cellulosic biohydrogen production

Among four cellulolytic microorganisms examined, Cellulomonas biazotea NCIM‐2550 can grow on various cellulosic substrates and produce reducing sugar. The activity of cellulases (endoglucanase, exoglucanase, and cellobiase), xylanase, amylase, and lignin class of enzymes produced by C. biazotea was mainly present extracellularly and the enzyme production was dependent on cellulosic substrates (carboxymethyl cellulose [CMC], sugarcane bagasse [SCB], and xylan) used for growth. Effects of physicochemical conditions on cellulolytic enzyme production were systematically investigated. Using MnCl₂ as a metal additive significantly induces the cellulase enzyme system, resulting in more reducing sugar production. The efficiency of fermentative conversion of the hydrolyzed SCB and xylan into clean H₂ energy was examined with seven H₂‐producing pure bacterial isolates. Only Clostridiumbutyricum CGS5 exhibited efficient H₂ production performance with the hydrolysate of SCB and xylan. The cumulative H₂ production and H₂ yield from using bagasse hydrolysate (initial reducing sugar concentration = 1.545 g/L) were approximately 72.61 mL/L and 2.13 mmol H₂/g reducing sugar (or 1.91 mmol H₂/g cellulose), respectively. Using xylan hydrolysate (initial reducing sugar concentration = 0.345 g/L) as substrate could also attain a cumulative H₂ production and H₂ yield of 87.02 mL/L and 5.03 mmol H₂/g reducing sugar (or 4.01 mmol H₂/g cellulose), respectively. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Optimization of carotenoid production by <Emphasis Type="Italic">Rhodotorula glutinis</Emphasis> using statistical experimental design

A two-step optimization strategy of statistical experimental design was employed to enhance carotenoid production from sugar cane molasses (SCM) in the yeast Rhodotorula glutinis. In the first step, a fractional factorial design was used to evaluate the impact of five fermentation factors (pH and concentrations of SCM, urea, KH₂PO₄, and NaCl). The results revealed that three factors (concentrations of SCM, urea, and KH₂PO₄) had a significant influence on biomass and carotenoid production. A face-centered central composite design was then used in the second step to optimize the three significant factors to further enhance the biomass yield and carotenoid production. Through this two-step optimization strategy, the carotenoid concentration could be increased from an average of 1.39 mg/l to an average of 3.46 mg/l, representing a 2.5-fold carotenoid production enhancement.     

Decolorization of beet molasses vinasse by Bacillus megaterium ATCC 14581

Beet molasses vinasse is formed in large quantities as a waste product of the distillery industry, where the molasses derived from sugar beet is used as a raw material. This vinasse has a dark color, low pH, and chelating properties. Many microorganisms have the ability to decolorize and biodegrade beet molasses vinasse. In this work, different cultivation parameters (the type of the bacterial culture; static or agitated) and medium components ((NH₄)₂SO₄, KH₂PO₄, yeast extract, glucose, and peptone, and the vinasse concentration) were evaluated utilizing Plackett-Burman design to identify the important factors influencing the vinasse decolorization by Bacillus megaterium ATCC 14581. The significant variables were selected as follows: (NH₄)₂SO₄, KH₂PO₄, glucose, and the concentration of vinasse. These four factors should be chosen as being promising for further optimization studies. The maximum color removal was 38%.     

Production of Alcaligenes xylosoxydans EMS33 in a Bench-scale Fermenter

Summary Optimization of medium composition and pH for chitinase production by the Alcaligenes xylosoxydans mutant EMS33 was carried out in the present study and the optimized medium composition and conditions were evaluated in a fermenter. The medium components screened initially using Plackett–Burman design were (NH₄)₂SO₄, MgSO₄ 7H₂O, KH₂PO₄, yeast extract, Tween 20 and chitin in shake flask experiments. The significant medium components identified by the Plackett–Burman method were MgSO₄ 7H₂O, Tween 20 and chitin. Central composite response surface methodology was applied to further optimize chitinase production. The optimized values of MgSO₄ 7H₂O, Tween 20, chitin and pH were found to be 0.6 g/l, 0.05 g/l, 11.5 g/l and 8.0, respectively. Chitinase and biomass production of Alcaligenes xylosoxydans EMS33, was studied in a 2-l fermenter containing (g/l): chitin, 11.5; yeast extract, 0.5; (NH₄)₂SO₄, 1; MgSO₄ 7H₂O, 0.6; KH₂PO₄, 1.36 and Tween 20, 0.05. The highest chitinase production was 54 units/ml at 60 h and pH 8.0 when the dissolved O₂ concentration was 60%, whereas the highest biomass production was achieved at 36 h and pH 7.5 without any dissolved O₂ control.     

Production of thuringiensin from Bacillus thuringiensis using a net-draft-tube,modified airlift reactor

A net-draft-tube, modified airlift reactor and a stirred-tank reactor were used for thuringiensin production by Bacillus thuringiensis subsp. darmstadiensis growing with various concentrations of molasses. The optimum concentration of molasses for thuringiensin production in both reactors was 15 g/l. There was a 6 h delay in sporulation in the modified airlift reactor compared with that in the stirred-tank reactor. Thuringiensin yield in the modified airlift reactor (2.2 g/l) was consequently higher than that in the stirred-tank reactor (1.1 g/l).