Improved Ethanol Production by Saccharomyces cerevisiae with EDTA,Ferrocyanide and Zeolite X Addition to Sugar Beet Molasses

The influence of ethylenediaminetetra acetic acid (EDTA), potassium ferrocyanide and zeolite X on ethanol production from sugar beet molasses by Saccharomyces cerevisiae was studied. For all of the three substances used, the effect was more pronounced when added to the fermentation medium rather than to the growth medium; 1·9 mmol dm⁻³ potassium ferrocyanide caused an increase in the final ethanol concentration of about 16·4% and 47·5% with respect to control culture on addition to growth and fermentation media respectively. The greatest stimulation in product yield was obtained with zeolite X introduced during the fermentation stage; 8·0 g dm⁻³ zeolite X increased ethanol concentration by 53·3%. © 1997 SCI.     

Batch and Continuous Production of Lactic Acid from Beet Molasses by Lactobacillus delbrueckii IFO 3202

Process variables were optimized for the production of lactic acid from pretreated beet molasses by Lactobacillus delbrueckii IFO 3202 for batch and continuous fermentations. In the batch fermentation, maximum yields (95·4% conversion, 77·1% effective) and maximum lactic acid volumetric productivity (4·83 g dm⁻³ h⁻¹) was achieved at 45°C, pH 6·0, 78·2 g dm⁻³ sugar concentration with 10 g dm⁻³ yeast extract. Various cheaper nitrogen sources were replaced with yeast extract on equal nitrogen bases in batch fermentation. Of all the nitrogen sources tested, yeast extract yielded the highest and malt sprouts yielded the second highest level of lactic acid. In the continuous fermentation, maximum lactic acid (4·15%) was obtained at a dilution rate of 0·1 h⁻¹. Maximum volumetric lactic acid productivity (11·20 g dm⁻³ h⁻¹) occurred at D = 0·5 h⁻¹ dilution rate. © 1997 SCI.     

Continuous ethanol production from carob pod extract by immobilized Saccharomyces cerevisiae in a packed-bed reactor

The continuous production of ethanol from carob pod extract by immobilized Saccharomyces cerevisiae in a packed-bed reactor has been investigated. At a substrate concentration of 150 g dm^?3, maximum ethanol productivity of 16 g dm^?3 h^?1 was obtained at D = 0·4 h^?1 with 62·3% of theoretical yield and 83·6% sugars′ utilization. At a dilution rate of 0·1 h^?1, optimal ethanol productivity was achieved in the pH range 3·5–5·5, temperature range 30–35·C and initial sugar concentration of 200 g dm^?3. Maximum ethanol productivity of 24·5 g dm^?3 h^?1 was obtained at D = 0·5 h^?1 with 58·8% of theoretical yield and 85% sugars′ utilization when non-sterilized carob pod extract containing 200 g dm^?3 total sugars was used as feed material. The bioreactor system was operated at a constant dilution rate of 0·5 h^?1 for 30 days without loss of the original immobilized yeast activity. In this case, the average ethanol productivity, ethanol yield (% of theoretical) and sugars′ utilization were 25 g dm^?3 h^?1, 58·8% and 85·5%, respectively.     

Batch production of L(+) lactic acid from whey by Lactobacillus casei (NRRL B‐441)

The effects of temperature, pH, and medium composition on lactic acid production by Lactobacillus casei were investigated. The highest lactic acid productivity values were obtained at 37 °C and pH 5.5. The productivity was 1.87 g dm^?3 h^?1 at 37 °C in shake flasks. In the fermenter, a productivity of 3.97 g dm^?3 h^?1 was obtained at pH 5.5. The most appropriate yeast extract concentration was 5.0 g dm^?3. Whey yielded a higher productivity value than the analytical lactose and glucose. Initial whey lactose concentration did not affect lactic acid productivity. MnSO₄ ·H₂O was necessary for lactic acid production by L casei from whey. Product yields were approximately 0.93 g lactic acid g lactose^?1. Copyright © 2004 Society of Chemical Industry     

Production and extractive biocatalysis of ethanol using microencapsulated yeast cells and lipase system

An extractive biocatalytic process for in situ ethanol recovery from fermentation broths of high glucose concentration (200 and 400 g dm^?3) was developed. In this system, ethanol was recovered by liquid-liquid extraction and enzymatic reaction. Oleic acid was used as extractant, allowing the esterification of ethanol catalysed by a lipase from Mucor miehei. The distribution coefficient of ethanol between the aqueous and the organic phases was improved tenfold by esterification. The better performance of the extractive fermentations of ethanol was obtained when co-immobilized Saccharomyces bayanus cells and a lipase from Mucor miehei in microemulsions of phosphatidylcholine were used. These results emphasize the important role of the surfactant in the protection of biocatalysts against organic phase toxicity.     

Continuous ethanol fermentation using immobilized yeast in a fluidized bed reactor

Continuous ethanol fermentation of glucose using fluidized bed technology was studied. Saccharomyces cerevisiae were immobilized and retained on porous microcarriers. Over two-thirds of the total reactor yeast cell mass was immobilized. Ethanol productivity was examined as dilution rate was varied, keeping all other experimental parameters constant. Ethanol yield remained high at an average of 0.36 g ethanol g^?1 glucose (71% of theoretical yield) as the dilution rate was increased stepwise from 0.04 h^?1 to 0.14 h^?1. At a dilution rate of 0.15 h^?1, the ethanol yield steeply declined to 0.22 g ethanol g^?1 glucose (44% of theoretical yield). The low maximum percentage of theoretical yield is primarily due to an extended mean cell residence time, and possibly due to the inhibitory effect of a high dissolved carbon dioxide concentration, enhanced by the probable intermittent levels of low pH in the reactor. Constant ethanol production was possible at a high glucose loading rate of 840 g dm^?3 day^?1 (attained at a dilution rate of 0.14 h^?1). Although the highest average ethanol concentration (97.14 g dm^?3) occurred at the initial dilution rate of 0.04 h^?1, the peak average ethanol production rate (2.87 g (g yeast)^?1 day^?1) was reached at a greater dilution rate of 0.11 ^h-1. Thus, the optimal dilution rate was determined to be between 0.11 h^?1 and 0.14 h^?1. Ethanol inhibition on yeast cells was absent in the reactor at average bulk-liquid ethanol concentrations as high as 97.14 g dm^?3. In addition, zero-order kinetics on ethanol production and glucose utilization was evident.     

Continuous production of thrombomodulin from a Pichia pastoris fermentation

A rotary membrane separation system was used in a continuous fermentation of Pichia pastoris with cell recycling to obtain high cell concentration and high thrombomodulin production. The dilution rates of this continuous fermentation were between 0·25 and 0·35 dm³ day⁻¹, and the production process was maintained for 10 days. Since cells were recycled and only part of liquid broth was taken from the system, a very high cell concentration level (248 g dm⁻³) was obtained. The peak protein expression level was at 72 h after methanol induction, was 300 mg dm⁻³ (3·6 × 10⁵ activity unit cm⁻³) and the total harvested supernatant was three times the working volume.     

Bacterial Bioconversion of Primary Aliphatic and Aromatic Alcohols into Acids: Effects of Molecular Structure and Physico-chemical Conditions

The biotransformation of four alcohol substrates (butanol, 2-methylbutanol, 3-methylbutanol and 2-phenylethanol) into their acids was studied using a strain of Acetobacter aceti. Bioconversion yields depended on the molecular structure of the alcohol. Biotransformation of high concentrations of alcohols was possible until the precursor reached an inhibiting concentration (3·8 g dm⁻³ for butanol and 3-methylbutanol, 4·2 g dm⁻³ for 2-methylbutanol). In contrast, biotransformation of 2-phenylethanol decreased when alcohol concentration was higher than 0·3 g dm⁻³. Dissolved oxygen concentrations and pH conditions of the medium were important factors in improving bioconversion. Transformation of 2-methylbutanol into the corresponding acid was increased when dissolved oxygen partial pressure increased from 60 to 80% and regulation at pH 6 allowed an increase in the production of butyric acid from butanol. © 1997 SCI.     

Cell recycle and broth reuse fermentation with cross-flow filtration and ion-exchange resin

A novel integrated fermentation system in which cross-flow filtration was coupled to an anion-exchange resin column was developed to achieve biomass recycle and broth reuse for lactic acid fermentation. An anion-exchange resin column was employed to recover lactic acid from the spent broth. The effluent was diluted with fresh medium, supplemented with glucose and nutrients. Spent broth was reused for three consecutive biomass recycle fermentations with no significant decrease in fermentation performance. The fermentation system enabled simultaneously high productivity of lactic acid (average value 7·75 g dm⁻³ h⁻¹ and total amount of lactic acid produced 85·21 g dm⁻³ after 11 h fermentation), high productivity of cells (average value 2·00 g dm⁻³ h⁻¹) and efficient utilization of medium (about 75% of the spent broth was reutilized). The system described may be applied to other organic fermentations subject to end-product inhibition.     

Biomass production by a thermotolerant yeast: Hansenula polymorpha

Biomass production at high temperature by Hansenula polymorpha as part of a lignocellulosic utilizing process was studied. Compromise growth conditions (45°C and pH = 4.8) with an eventual saccharification step were established. The effects of stirring rate and initial glucose concentration on biomass yield coefficient, volumetric productivity and maximal cell density were determined. Process optimization led to a fed-batch fermentation process: high yield (0.63 g dry cell g^?1 glucose), volumetric productivity (1.3 g dry cell dm^?3 h^?1) and cell concentration (60 g dry cell dm^?3) were obtained. At these conditions, significant arabitol excretion (18 g dm^?3) as a unique by-product associated with cell mass production was obtained, making more interesting a high temperature operating process.     

Effect of extraction and enzymatic esterification of ethanol on glucose consumption by two Saccharomyces cerevisiae strains: a comparative study

Extractive alcoholic fermentations of high glucose concentrations (300 and 400 g dm^?3) using a flocculent (saké) and a non‐flocculent (DER24) Saccharomyces cerevisiae strain were compared. The introduction of a Rhizomucor miehei lipase, in the extractive fermentations of 300 g dm^?3 of glucose, increased the ethanol extraction due to its esterification with oleic acid, allowing complete glucose consumption at an organic solvent/fermentation medium phase ratio of 1. In these conditions, an increase of ethanol yield was observed. Total glucose consumption was also obtained in enzymatic extractive fermentations of 400 g dm^?3 of glucose, but only when oleic acid was added at the exponential growth phase. From the comparison of the extractive fermentation performances obtained using the two yeast strains it was observed that the flocculent strain led to a lower glucose metabolisation rate. This behaviour was related to the highest diffusional limitations that occur in the presence of flocs. The developed processes showed that the association of alcoholic fermentation with enzymatic extraction led to the reduction of inhibitory effects as well as to the simultaneous production of fatty esters which are compounds with several commercial applications. © 2001 Society of Chemical Industry     

Factors affecting silver biosorption by an industrial strain of Saccharomyces cerevisiae

Factors affecting silver biosorption by Saccharomyces cerevisiae biomass, obtained as a waste product from industry, were examined. Maximum removal of silver from solution was achieved within 5 min. Increasing the concentration of biomass in experimental flasks from 1 to 8 mg cm⁻³ decreased both silver accumulation, from 224·7 to 89·5 μmol Ag g⁻¹ dry wt, and associated H⁺ ion release, from 109·4 to 31·7 μmol H⁺ g⁻¹ dry wt. The presence of 1·0 mol dm⁻³ cadmium or methionine decreased silver biosorption by 40% and 93% respectively. Boiling in 100 mmol dm⁻³ NaOH or 10 mmol dm⁻³ sodium dodecyl sulphate decreased silver biosorption by 54% and 25% respectively. A temperature increase from 4°C to 55°C decreased silver biosorption by 9%. The metabolic state of the yeast had no effect on silver biosorption. Decreasing the pH of the silver solution caused a reduction in metal removal by the biomass.     

Production of lactic acid from beet molasses by calcium alginate immobilized Lactobacillus delbrueckii IFO 3202

Lactic acid was produced from pretreated beet molasses by the homofermentative organism Lactobacillus delbrueckii subsp delbrueckii IFO 3202 entrapped in calcium alginate gel using batch, repeated batch and continuous fermentation systems. In batch fermentation studies successful results were obtained with 2.0–2.4 mm diameter beads prepared from 2% sodium alginate solution. The highest effective yield (82.0%) and conversion yield (90.0%) were obtained from substrate concentrations of 52.1 and 78.2 g dm⁻³ respectively. The gel beads produced lactic acid for 14 consecutive batch fermentations without marked activity loss and deformation. In the continuous fermentation, the highest lactic acid (4.22%) was obtained at a dilution rate of 0.1 h⁻¹ while the highest productivity (13.92 g dm⁻³ h⁻¹) was obtained at a dilution rate of 0.4 h⁻¹. © 1999 Society of Chemical Industry     

Production of optically pure L-alanine by immobilized Pseudomonas sp. BA2 cells

The conditions for immobilizing the new L -aminoacylase-producing bacterial strain, Pseudomonas sp. BA2, by entrapment in κ-carrageenan gel, were investigated. The optimal gel concentration and cell load were determined. The addition of CoCl₂ and N-acetyl-L -alanine to the immobilizing matrix enhanced L -aminoacylase activity. The enzymatic properties of immobilized Pseudomonas sp. BA2 were investigated. Enzyme activity in immobilized cells was optimal at a pH of 6·5 using 0·15 mol dm⁻³ Tris–maleate buffer at 45°C. The presence of 0·7 mmol dm⁻³ CoCl₂ in the enzymatic reaction mixture improved L -aminoacylase activity. The immobilized cell preparation was used for the production of L -alanine from N-acetyl-DL -alanine in a batch reactor. Conversions of 100% were obtained using substrate concentrations ranging from 20 to 200 mmol dm⁻³. The reactor production was 0·74 mol h⁻¹ g cell⁻¹ dm⁻³ which is noticeably higher than that previously reported in the literature. © 1998 Society of Chemical Industry     

Purification of the Penicillium citrinum Lipase Using AOT Reversed Micelles

This work describes the extraction and back-extraction of a lipase from crude extract of Penicillium citrinum using AOT reversed micelles in isooctane. The effect of pH, ionic strength, AOT concentration on the protein forward and backward transfer at 20°C was studied. The maximum protein forward extraction (32·0%) was achieved at pH 4·0 with a 50 mmol dm⁻³ acetate buffer containing 100 mmol dm⁻³ KCl and 100 mmol dm⁻³ AOT in isooctane. Proteins were back-extracted (82·7%) to a new aqueous phase containing 100 mmol dm⁻³ pH 8·0 phosphate buffer and 1000 mmol dm⁻³ KCl. No enzyme activity could be detected either in the micellar phase or in the aqueous phase after protein back-extraction. However, the lipolytic activity was recovered after hydrophobic interaction chromatography on a Phenyl Superose column. The yield obtained for the overall process was 68% for activity, 26·4% for protein recovery and the purification factor was 810-fold. A single protein band at 33000 Da was obtained for SDS–PAGE analysis for the recovered and purified enzyme. © 1997 SCI.     

Technological and kinetic aspects of sublethal acid toxicity in microbial gum production

Chemostat culture of Xanthomonas campestris were obtained at a dilution rate of 0·05 h⁻¹ and the normal feed then supplemented with 0·58 and 1·74 mmol dm⁻³ isobutyric acid (IBA). Data revealed that the organism responded to sublethal acid stress by overproducing xanthan. The acid additions led to transient zones in the continuous cultivation profiles. By adding feed containing 1·74 mmol dm⁻³ IBA, volumetric growth rate immediately decreased from 0·059 to 0·026 g dm⁻³ h⁻¹ whereas the specific xanthan formation rate increased from 0·23 g g⁻¹ biomass h⁻¹ to a maximum 0·65 g g⁻¹ biomass h⁻¹ (with 1·0 mmol dm⁻³ IBA addition), before decreasing as the concentration of acid attained that of the feed. By monitoring the outlet CO₂ in parallel with biomass and polysaccharide levels in the IBA fermentation a 10% diversion of the total carbon flux from biomass synthesis to xanthan biosynthesis was detected. A consistent pattern of variation in activity was detected in enzymes of intermediary metabolism, suggesting an action at the regulatory level. Enhanced activities of carbon catabolism and xanthan anabolic reactions (phosphomannose isomerase) were observed in the presence of the acid. Batch experiments carried out in the pres-ence of IBA gave results which correlated with the undissociated acid form con-centration. An undissociated acid fraction of 6·5×10⁻³ mmol dm⁻³ was calculated in a set of flasks under the same conditions and a statistically vali-dated 12% increase in xanthan production was found. The maximum activation was determined to be below 1·1×10⁻² mmol dm⁻³ when a 58% specific xanthan production rate increase occurred in parallel with a 35% decrease in biomass concentration.     

Production of Hexanoic Acid by Free and Immobilised Cells of Megasphaera elsdenii: Influence of in-situ Product Removal Using Ion Exchange Resin

The objective of this work was to improve the production of hexanoic acid by the anaerobic rumen bacterium, Megasphaera elsdenii, using product removal and immobilised cell approaches. Hexanoic acid, the major product of glucose metabolism by M. elsdenii strain ATCC25940, was produced at concentrations of 2–3 g dm⁻³ in stirred batch cultures. With pH controlled manually at 7, maximum concentrations of hexanoic acid increased to 6–8 g dm⁻³ with yields (g product per g glucose used) of approximately 30%. When an anion exchange resin, Amberlite IRA 400, was added during early stages of culture to minimise product inhibition, growth was not impaired and cell lysis, which was commonly seen during the stationary phase in control fermentations, was prevented. The presence of resin in pH-controlled, stirred batch fermentations increased the rate of glucose consumption and doubled hexanoic acid productivity: the equivalent of 11 g dm⁻³ of hexanoic acid was made with an estimated yield of up to 39%. Cells were immobilised successfully in κ-carrageenan and, when cell densities in inocula were sufficiently high, rates of glucose consumption and product formation were similar to free cells. Including resin in cultures of immobilised cells had effects similar to those above. Using a fed-batch mode with immobilised cells cultured in the presence of resin further increased final concentrations of hexanoic acid (up to 19 g dm⁻³) but yields were lower (20–30%) and productivity did not increase. These results show that production of volatile fatty acids can be improved significantly by product stripping onto an anion exchange resin. © 1997 SCI.     

The effect of D‐glucose on milk clotting activity of Mucor miehei in a chemostat with biomass retention

In this study microbial production of rennin from a commercial strain of Mucor miehei (NRRL 3420) has been accomplished in a continuously fed fermenter. The effects of feed D ‐glucose concentration (2.5–30 g dm⁻³) on milk clotting activity and on other system variables were investigated at optimum mixing and dilution rates of 400 rpm and 0.0052 h⁻¹, respectively, without pH control. Maximum milk clotting activity (1.24 IU cm⁻³) occurred when the feed D ‐glucose concentration was 7.5 g dm⁻³. Enzyme production continued for 500 h, producing a total milk clotting activity of 10 230 IU. At the maximum milk clotting activity point, total protein concentration, milk clotting and proteolytic activities were analysed and compared with those of a commercial rennet. The ratio of milk clotting activity to proteolytic activity, and specific milk clotting activity were determined as 1.55 × 10⁻³ IU PU⁻¹ and 5.28 IU mg⁻¹ medium protein, respectively, denoting similar characteristics to a commercial rennet after concentration of the fermentation medium. © 1999 Society of Chemical Industry     

Production of fructooligosaccharides by β‐fructofuranosidase from Aspergillus sp 27H

β‐fructofuranosidase (EC 3.2.1.26) from Aspergillus sp 27H isolated from soil was investigated for production of fructooligosaccharides (FOS) using whole cells. It possesses hydrolytic and transfructosylating activities that can be altered by modifying the reaction conditions. The optimal conditions for the transfructosylating activity occur in the pH range 5.5–6.0 and at 60 °C, while hydrolytic activity was highest at pH 4.0 and 55 °C. At low sucrose concentration (10 g dm^?3) there was rapid conversion of sucrose to glucose and fructose and very low concentrations of FOS were obtained. However, at sucrose concentrations higher than 216 g dm^?3 the concentrations of hydrolysis products were reduced. Under the following conditions: pH 5.5, temperature 40 °C, sucrose concentration 615 g dm^?3 and enzyme concentration 20β‐fructofuranosidase units g^?1 of sucrose, the FOS concentration reached a maximum value of 376 g dm^?3 (234 g dm^?3 1‐kestose and 142 g dm^?3 nystose) and the proportion of FOS in the solids in the reaction mixture was 600–620 g kg^?1 at 6 h. These results suggest that β‐fructofuranosidase from Aspergillus sp 27H could be an appropriate enzyme for the commercial production of FOS. Copyright © 2004 Society of Chemical Industry     

Kinetic study on ethanol production using Saccharomyces cerevisiae ITV‐01 yeast isolated from sugar cane molasses

BACKGROUND: Bio‐ethanol production from renewable sources, such as sugar cane, makes it a biofuel that is both renewable and environmentally friendly. One of the strategies to reduce production costs and to make ethanol fuel economically competitive with fossil fuels could be the use of wild yeast with osmotolerance, ethanol resistance and low nutritional requirements. The aim of this work was to investigate the kinetics of ethanol fermentation using Saccharomyces cerevisiae ITV‐01 yeast strain in a batch system at different glucose and ethanol concentrations, pH values and temperature in order to determine the optimum fermentation conditions. RESULTS: This strain showed osmotolerance (its specific growth rate (µ_max) remained unchanged at glucose concentrations between 100 and 200 g L^?1) as well as ethanol resistance (it was able to grow at 10% v/v ethanol). Activation energy (Ea) and Q₁₀ values calculated at temperatures between 27 and 39 °C, pH 3.5, was 15.6 kcal mol^?1 (with a pre‐exponential factor of 3.8 × 10¹² h^?1 (R² = 0.94)) and 3.93 respectively, indicating that this system is biologically limited. CONCLUSIONS: The optimal conditions for ethanol production were pH 3.5, 30 °C and initial glucose concentration 150 g L^?1. In this case, a maximum ethanol concentration of 58.4 g L^?1, ethanol productivity of 1.8 g L^?1 h^?1 and ethanol yield of 0.41 g g^?1 were obtained. Copyright © 2010 Society of Chemical Industry