High productivity ethanol fermentations with Zymomonas mobilis using continuous cell recycle

Summary Cell recycle studies have been carried out with a strain of Zymomonas mobilis selected for its improved ethanol tolerance and faster rates of glucose uptake and ethanol production. As part of the investigation a capilliary cross-flow microfiltration unit with polyamide membranes has been evaluated in view of its potential advantages (low cost and ability to withstand repeated cleaning with caustic soda). The results demonstrate that ethanol concentrations of 60–65g/l can be sustained at productivities ranging from 120–200g/l/h.     

Kinetics of ethanol fermentations in membrane cell recycle fermentors

Ethanol fermentation by yeast was carried out in a cell filtration recycle system with a hollow-fiber membrane filter. Maximum biomass concentrations up to 210 g dry wt/L were obtained, but in normal operation concentrations they were between 100 and 150 g/L. The ethanol productivity using 14% glucose feed was 85 g/L h, with an ethanol concentration of 65 g/L and an ethanol yield of over 90%. The ethanol productivity and yeast growth rate decreased as the cell concentration increased beyond a certain level. The cell mass in the reactor was maintained by a proper manipulation of diluticn rate and bleed ratio depending on the growth rate.     

Continuous ethanol production using cell recycle with a settler

Summary A system for a high-density culture of Zymomonas mobilis was tested using a reactor with cell recycle by means of a simple settler. Growth-limiting conditions were created by raising the temperature and lowering the amount of yeast-extract. In this case the biomass production decreased, while the specific ethanol productivity did not change markedly. Under these conditions we succeeded in creating a system with a productivity of 40.5 g.1^-1.h^-1. The choice for optimal design of the settler and optimal conditions has to be made by optimization taking into account economic and whole process considerations.     

Improvements in ethanol concentration and fermentor ethanol productivity in yeast fermentations using whole soy flour in batch,and continuous recycle systems

Summary Ethanol concentrations and fermentor productivities were increased 20.2 and 15.5% at 90 and 95% recycle, respectively, when whole soy flour was added to the feed (2 g/ at 90% recycle, and 1 g/ at 95% recycle) of a continuous yeast fermentation system with recycle of cells and soy flour (soy flour concentrations were 2% at steady state in the fermentor) by UF membranes as compared to controls. The improvements were primarily due to increases in cell concentrations. Similar results were obtained for batch cultures.     

Production of xylitol in cell recycle fermentations of Candida tropicalis

Xylitol was produced a in two-substrate, batch fermentation with cell recycling of Candida tropicalis ATCC 13803. A series of cell-recycle experiments showed that the feeding of xylose, glucose and yeast extract in the xylitol production phase was most effective in enhancing xylitol productivity. The optimized cell recycle fermentation resulted in 0.82 g xylitol/g xylose yield, 4.94 g xylitol l^–1 h^–1 productivity, and final xylitol concentration of 189 g l^–1. These results were 1.3 times higher in volumetric xylitol productivity and 2.2 times higher in final product concentration compared with the corresponding values of the optimized two-substrate batch culture.     

Enhanced product formation in continuous fermentations with microbial cell recycle*

The effect of partial recycle of microbial cells on the operation of a chemostat has been investigated for two fermentations. Stable steady states without partial cell recycle were obtained for the conversion of D -sorbitol to L -sorbose by Gluconobacter oxydans subsp. suboxydans 1916B and for the conversion of glucose to 2-ketogluconic acid by Serratia marcescens NRRL B-486. The employment of partial cell recycle dramatically increased product formation rates for both fermentations.     

Fermentation of cellodextrins to ethanol using mixed-culture fermentations

The potential for enhancing ethanol production from cellodextrins by employing mixed-culture (Candida wickerhamii-Saccharomyces cerevisiae) fermentations was investigated. Initially, ethanol production was monitored in fermentation medium containing 50 g/L glucose plus 45 g/L cellobiose. Inoculum levels and times of inoculum addition were varied. Of the conditions tested, the most rapid rates of ethanol formation occurred in fermentations in which either C. wickerhamii and S. cerevisiae were coinoculated at a ratio of 57 : 1 cell/mL or in fermentations in which a 10-fold-greater S. cerevisiae inoculum was added to a pure culture C. wickerhamii fermentation after 1 day incubation. These conditions were used to attempt to enhance fermentations in which cellodextrins produced by trifluoroacetic acid hydrolysis of cellulose served as the sole carbon source. Cellodextrins that were not further purified after cellulose hydrolysis contained compounds that were slightly inhibitory to C. wickerhamii. In this case the mixed-culture fermentations produced 12-45% more ethanol than a pure culture C. wickerhamii fermentation. However, if the substrate was treated with Darco G-60 charcoal, the toxic materials were apparently removed and the pure culture C. wickerhamii fermentations performed as well as the mixed-culture fermentations.     

Continuous and biomass recycle fermentations of Clostridium acetobutylicum

The availability and demand of biosynthetic energy (ATP) is an important factor in the regulation of solvent production in steady state continuous cultures of Clostridium acetobutylicum. The effect of biomass recycle at a variety of dilution rates and recycle ratios under both glucose and non-glucose limited conditions on product yields and selectivities has been investigated. Under conditions of non-glucose limitation, when the ATP supply is not growth-limiting, a lower growth rate imposed by biomass recycle leads to a reduced demand for ATP and substantially higher acetone and butanol yields. When the culture is glucose limited, however, biomass recycle results in lower solvent yields and higher acid yields.List of Symbols A ₆₀₀ absorbance at 600 nm - ATP adenosine triphosphate - C _imol/dm³ concentration of componenti in the fermentor - C _i ⁰ mol/dm³ concentration of componenti in the feed - D h^–1 dilution rate - F dm³/h feed flow rate - FdH₂ ferredoxin, reduced form - NAD nicotinamide adenine dinucleotide, oxidized form - NADH nicotinamide adenine dinucleotide, reduced form - NfF mmol/g/h NADH produced from oxidation of FdH₂ per unit biomass per unit time - P dm³/h filtrate flow during biomass recycle operation - PCRP C-mole carbon per C-mole glucose utilized percent of (substrate) carbon recovered in products - R recycle ratio,P/F - SPR mmol/g/h specific production rate - X _imol product/100 mol glucose utilized product yield - Y _ATP g biomass/mol ATP biomass yield on ATP - Y _GLU g biomass/mol glucose biomass yield on glucose - Y _ig biomass/mol biomass yield on nutrienti - h^–1 specific growth rate     

Continuous and biomass recycle fermentations of Clostridium acetobutylicum

Using experimental data from continuous cultures of Clostridium acetobutylicum with and without biomass recycle, relationships between product formation, growth and energetic parameters were explored, developed and tested. For glucose-limited cultures the maintenance models for, the Y _ATP and biomass yield on glucose, and were found valid, as well as the following relationships between the butanol (Y _B/G) or butyrate (Y _BE/G) yields and the ATP ratio (R _ATP, an energetic parameter), Y _B/G =0.82-1.35 R _ATP, Y _BE/G =0.54 + 1.90 R _ATP. For non-glucose-limited cultures the following correlations were developed, Y _B/G =0.57-1.07 , Y _B/G =0.82-1.35 R _ATPATP and similar equations for the ethanol yield. All these expressions are valid with and without biomass recycle, and independently of glucose feed or residual concentrations, biomass and product concentrations. The practical significance of these expressions is also discussed.List of Symbols D h^–1 dilution rate - m _e mol g^–1 h^–1 maintenance energy coefficient - m _G mol g^–1 h^–1 maintenance energy coefficient - R biomass recycle ratio, (dimensionless) - R _ATP ATP ratio (eqs.(5), (10) and (11)), (dimensionless) - X kg/m³ biomass concentration - Y _ATP g biomass per mol ATP biomass yield on ATP - Y _ATP ^max g biomass per mol ATP maximum Y _ATP - Y _A/G mol acetate produced per mol glucose consumed molar yield of acetate - y _an/g mol acetone produced per mol glucose consumed molar yield of acetone - Y _B/G mol butanol produced per mol glucose consumed molar yield of butanol - y _be/g mol butyrate produced per mol glucose consumed molar yield of butyrate - Y _E/G mol ethanol produced per mol glucose consumed molar yield of ethanol - Y _X/G g biomass per mol glucose consumed biomass yield on glucose - Y _ATP ^max g biomass per mol maximum Y _X/G glucose consumed - h^–1 specific growth rate     

High cell density ethanol fermentation in an upflow packed-bed cell recycle bioreactor

An upflow packed-bed cell recycle bioreactor (IUPCRB) is proposed for obtaining a high cell density. The system is comprised of a stirred tank bioreactor in which cells are retained partially by a packed-bed. A 1.3 cm (ID) × 48 cm long packed-bed was installed inside a 2 L bioreactor (working volume 1 L). Continuous ethanol fermentation was carried out using a 100 g/L glucose solution containing Saccharomyces cerevisiae (ATCC 24858). Cell retention characteristics were investigated by varying the void fraction (VF) of the packed bed by packing it with particles of 0.8∼2.0 mm sized stone, cut hollow fiber pieces, ceramic, and activated carbon particles. The best results were obtained using an activated carbon bed with a VF of 30∼35%. The IUPCRB yielded a maximum cell density of 87 g/L, an ethanol concentration of 42 g/L, and a productivity of 21 g/L/h when a 0.5 h⁻¹ dilution rate was used. A natural bleeding of cells from the filter bed occurred intermittently. This cell loss consisted of an average of 5% of the cell concentration in the bioreactor when a high cell concentration (approximately 80 g/L) was being maintained.     

Continuous recombinant bacterial fermentations utilizing selective flocculation and recycle.

Selective recycle has successfully been used to maintain an unstable plasmid-bearing bacterial strain as dominant in a continuous reactor, whereas the culture reverts to 100% segregant cells when selective recycle is not used. The plasmid-bearing strain is slower growing and flocculent; however, when the cells lose their plasmid, the resulting segregant cells are nonflocculent and grow at a faster rate due to their decreased metabolic burden. Both types of cells exit a chemostat and enter an inclined settler where the flocculent plasmid-bearing cells are separated from the nonflocculent segregant cells by differential sedimentation. The underflow from the cell separator, which is enriched with plasmid-bearing cells, is recycled back to the chemostat, while the segregant cells are withdrawn off the top of the settler and discarded. The experimental results agree well with selective recycle reactor theory. On the basis of the theory, a criterion is presented that has been shown to successfully predict whether or not a selective recycle reactor can maintain a plasmid-bearing strain.     

Microbial contamination of fuel ethanol fermentations

Microbial contamination is a pervasive problem in any ethanol fermentation system. These infections can at minimum affect the efficiency of the fermentation and at their worse lead to stuck fermentations causing plants to shut down for cleaning before beginning anew. These delays can result in costly loss of time as well as lead to an increased cost of the final product. Lactic acid bacteria (LAB) are the most common bacterial contaminants found in ethanol production facilities and have been linked to decreased ethanol production during fermentation. Lactobacillus sp. generally predominant as these bacteria are well adapted for survival under high ethanol, low pH and low oxygen conditions found during fermentation. It has been generally accepted that lactobacilli cause inhibition of Saccharomyces sp. and limit ethanol production through two basic methods; either production of lactic and acetic acids or through competition for nutrients. However, a number of researchers have demonstrated that these mechanisms may not completely account for the amount of loss observed and have suggested other means by which bacteria can inhibit yeast growth and ethanol production. While LAB are the primary contaminates of concern in industrial ethanol fermentations, wild yeast may also affect the productivity of these fermentations. Though many yeast species have the ability to thrive in a fermentation environment, Dekkera bruxellensis has been repeatedly targeted and cited as one of the main contaminant yeasts in ethanol production. Though widely studied for its detrimental effects on wine, the specific species–species interactions between D. bruxellensis and S. cerevisiae are still poorly understood.     

Rapid immunoassay technique for process monitoring of animal cell fermentations.

A calibration and quality control technique suited to process monitoring with immunoassay is demonstrated. The particle concentration fluorescence immunoassay (PC-FIA) is shown to provide a sensitive and rapid method for the quantification of specific biomolecules in cell cultures. Smoothing of linear calibration parameters is performed by forming weighted averages of standard points as the run progresses. These estimates are then used to determine slope and intercept values for improved calibration. The nonuniformity of the fluorescent signal variance is also considered, and a weight model is developed to describe the relationship between signal fluorescence and signal variance for weighted linear curve fitting. Pooling calibration results over the process run improves overall assay performance as determined by using standard control chart analysis. This method is suitable for semicontinuous monitoring of animal cell fermentations and has been used here to measure cell-associated and culture supernatant concentrations of monoclonal antibody (Ab) from hybridoma cells. The cell-associated Ab concentration correlates with cell-specific production rate. Assay times on the order of 10 min for supernatant and 25-30 min for cell-associated Ab concentrations can be achieved, making this procedure suitable for process monitoring and control. Under these conditions the assay has a detection limit of approximately 10 ng/mL, providing a sensitive and specific method for the quantification of cell culture constituents.     

Fermentation coupled with microfiltration: kinetics of ethanol fermentation with cell recycle

Cell recycle by microfiltration was used in yeast alcoholic fermentation in continuous operation. Toxins were proved to be washed by increasing dilution rate. — Specific ethanol production rate followed an exponential inhibition equation, which is function of both biomass concentration and dilution rate. — Productivity is shown to be 40 times greater than in conventional continuous operation.     

New cell recycle ethanol fermentation with periodic cleaning of filter with gas

High ethanol productivities were obtained by cell recycle cultures of yeast and bacterial strains at a dry cell concentration of 200 kg cells m^–3 using a new membrane bioreactor system. The filtration rates of the cultures were stabilized by removing the microbial cake on the filter with periodic back flows of the fermentation gas through the filter. For instance, the filtration flux of 0.023 m³m^–2h^–1 was maintained for 30 h with the periodic cleaning of the filter, whereas it decreased at a half time of 2 h without the cleaning. Ethanol productivity, ethanol concentration and filtration flux attained were: 68.7 kg/(m³ · h), 62.7 kg/m³ and 0.029 m³m^–2h^–1 for Saccharomyces carlsbergensis, LAM1068, the respective values for Zymomonas mobilis, ZM4, were: 93.7, 33 5 and 0.074.     

Role of water activity in ethanol fermentations

A separate role for water activity in the conversion of sugars to ethanol by two strains of yeast is identified. During fermentation of both single and mixed sugar substrates, the water activity was shown to remain constant during the logarithmic growth phase. This is despite the changes in concentration of substrates and product, the constancy reflecting the fact that the greater influence of ethanol on the solution activity is counterbalanced, in the early stages of the fermentation, by its low yield. The end of the log phase of growth coincides with the start of a period of gradually decreasing water activity. For the more ethanol-tolerant strain UQM66Y, growth was found to cease at a constant value of water activity while that for the less tolerant strain UQM70Y depended on both ethanol concentration and water activity. It is argued that water activity is a more appropriate variable than ethanol concentration for describing some of the nonspecific inhibitory effects apparent in ethanol fermentations. A straightforward method for the calculation of water activity during such fermentations based on the use of solution osmolality is presented.     

Very high ethanol productivity in an innovative continuous two-stage bioreactor with cell recycle

The performance of an innovative two-stage continuous bioreactor with cell recycle—potentially capable of giving very high ethanol productivity—was investigated. The first stage was dedicated to cell growth, whereas the second stage was dedicated to ethanol production. A high cell density was obtained by an ultrafiltration module coupled to the outlet of the second reactor. A recycle loop from the second stage to the first one was tested to improve cell viability and activity. Cultivations of Saccharomyces cerevisiae in mineral medium on glucose were performed at 30°C and pH 4. At steady state, total biomass concentrations of 59 and 157 gDCW l⁻¹ and ethanol concentrations of 31 and 65 g l⁻¹ were obtained in the first and second stage, respectively. The residual glucose concentration was 73 g l⁻¹ in the first stage and close to zero in the second stage. The present study shows that a very high ethanol productivity (up to 41 g l⁻¹ h⁻¹) can indeed be obtained with complete conversion of the glucose and with a high ethanol titre (8.3°GL) in the two-stage system.     

Replicative inactivation and metabolic inhibition in yeast ethanol fermentations

Summary The effects of ethanol on the growth rates of twoSaccharomyces yeast strains were measured during normal batch fermentative growth and compared with those measured by initial rate studies. In the light of previous work, which has highlighted the loss of cell replicative ability caused by ethanol, the results imply that the observed reduction in growth rate reflects a mixture of true inhibition and replicative inactivation.