Improved ethanol production from xylose with glucose isomerase and Saccharomyces cerevisiae using the respiratory inhibitor azide

Summary Ethanol was produced from xylose, using the enzyme glucose isomerase (xylose isomerase) and Saccharomyces cerevisiae. The influence of aeration, pH, enzyme concentration, cell mass and the concentration of the respiratory inhibitor sodium azide on the production of ethanol and the formation of by-products was investigated. Anaerobic conditions at pH 6.0, 10 g/l enzyme, 75 g/l dry weight cell mass and 4.6 mM sodium azide were found to be optimal. Under these conditions theoretical yields of ethanol were obtained from 42 g/l xylose within 24 hours.In a fed-batch culture, 62 g/l ethanol was produced from 127 g/l xylose with a yield of 0.49 and a productivity of 1.35 g/l·h.     

Xylitol production from D-xylose byCandida guillermondii: Fermentation behaviour

Summary The ability ofCandida guillermondii to produce xylitol from xylose and to ferment individual non xylose hemicellulosic derived sugars was investigated in microaerobic conditions. Xylose was converted into xylitol with a yield of 0,63 g/g and ethanol was produced in negligible amounts. The strain did not convert glucose, mannose and galactose into their corresponding polyols but only into ethanol and cell mass. By contrast, fermentation of arabinose lead to the formation of arabitol. On D-xylose medium,Candida guillermondii exhibited high yield and rate of xylitol production when the initial sugar concentration exceeded 110 g/l. A final xylitol concentration of 221 g/l was obtained from 300 g/l D-xylose with a yield of 82,6% of theoretical and an average specific rate of 0,19 g/g.h.Nomenclature Q_p average volumetric productivity of xylitol (g xylitol/l per hour) - q_p average specific productivity of xylitol (g xylitol/g of cells per hour) - So initial xylose concentration (g/l) - tf incubation time (hours) - Y_P/S xylitol yield (g of xylitol produced/g of xylose utilized) - Y_E/S ethanol yield (g of ethanol produced/g of substrate utilized) - Y_X/S cells yield (g of cells/g of substrate utilized) - specific growth rate coefficient (h^–1) - _max maximum specific growth rate coefficient (h^–1)     

Repeated-batch fermentations of xylose and glucose-xylose mixtures using a respiration-deficient Saccharomyces cerevisiae engineered for xylose metabolism

Xylose-fermenting Saccharomyces strains are needed for commercialization of ethanol production from lignocellulosic biomass. Engineered Saccharomyces cerevisiae strains expressing XYL1, XYL2 and XYL3 from Pichia stipitis, however, utilize xylose in an oxidative manner, which results in significantly lower ethanol yields from xylose as compared to glucose. As such, we hypothesized that reconfiguration of xylose metabolism from oxidative into fermentative manner might lead to efficient ethanol production from xylose. To this end, we generated a respiration-deficient (RD) mutant in order to enforce engineered S. cerevisiae to utilize xylose only through fermentative metabolic routes. Three different repeated-batch fermentations were performed to characterize characteristics of the respiration-deficient mutant. When fermenting glucose as a sole carbon source, the RD mutant exhibited near theoretical ethanol yields (0.46 g g(-1)) during repeated-batch fermentations by recycling the cells. As the repeated-batch fermentation progressed, the volumetric ethanol productivity increased (from 7.5 to 8.3 g L(-1)h(-1)) because of the increased biomass from previous cultures. On the contrary, the mutant showed decreasing volumetric ethanol productivities during the repeated-batch fermentations using xylose as sole carbon source (from 0.4 to 0.3 g L(-1)h(-1)). The mutant did not grow on xylose and lost fermenting ability gradually, indicating that the RD mutant cannot maintain a good fermenting ability on xylose as a sole carbon source. However, the RD mutant was capable of fermenting a mixture of glucose and xylose with stable yields (0.35 g g(-1)) and productivities (0.52 g L(-1)h(-1)) during the repeated-batch fermentation. In addition, ethanol yields from xylose during the mixed sugar fermentation (0.30 g g(-1)) were higher than ethanol yields from xylose as a sole carbon source (0.21 g g(-1)). These results suggest that a strategy for increasing ethanol yield through respiration-deficiency can be applied for the fermentation of lignocellulosic hydrolyzates containing glucose and xylose.     

Adaptation of a recombinant xylose-utilizing Saccharomyces cerevisiae strain to a sugarcane bagasse hydrolysate with high content of fermentation inhibitors

Adaptation of a xylose-utilizing genetically engineered strain of Saccharomyces cerevisiae to sugarcane bagasse hydrolysates by cultivation during 353h using medium with increasing concentrations of inhibitors, including phenolic compounds, furaldehydes and aliphatic acids, led to improved performance with respect to ethanol production. The remaining xylose concentration in the medium at the end of the cultivation was 5.2g l(-1), while it was 11gl(-1) in the feed, indicating that approximately half of the xylose was consumed. The performance of the adapted strain was compared with the parental strain with respect to its ability to ferment three bagasse hydrolysates with different inhibitor concentration. The ethanol yield after 24h of fermentation of the bagasse hydrolysate with lowest inhibitor concentration increased from 0.18gg(-1) of total sugar with the non-adapted strain to 0.38gg(-1) with the adapted strain. The specific ethanol productivity increased from 1.15g ethanol per g initial biomass per h with the non-adapted strain to 2.55gg(-1) h(-1) with the adapted strain. The adapted strain performed better than the non-adapted also in the two bagasse hydrolysates containing higher concentrations of inhibitors. The adapted strain converted the inhibitory furaldehydes 2-furaldehyde (furfural) and 5-hydroxymethyl-2-furaldehyde (HMF) at a faster rate than the non-adapted strain. The xylose-utilizing ability of the yeast strain did not seem to be affected by the adaptation and the results suggest that ethanol rather than xylitol was formed from the consumed xylose.     

A novel circulating loop bioreactor with cells immobilized in loofa (<Emphasis Type="Italic">Luffa cylindrica</Emphasis>) sponge for the bioconversion of raw cassava starch to ethanol

A circulating loop bioreactor (CLB) with cells immobilized in loofa sponge was constructed for simultaneous aerobic and anaerobic processes. The CLB consists of an aerated riser and a non-aerated downcomer column connected at the top and bottom by cylindrical pipes. Ethanol production from raw cassava starch was investigated in the CLB. Aspergillus awamori IAM 2389 and Saccharomyces cerevisiae IR2 immobilized on loofa sponge were placed, respectively, in the aerated riser column and non-aerated downcomer column. Both alpha-amylase and glucoamylase activities increased as the aeration rate was increased. Ethanol yield and productivity increased with an increase in the aeration rate up to 0.5 vvm, but decreased at higher aeration rates. The CLB was operated at an aeration rate of 0.5 vvm for more than 600 h, resulting in an average ethanol productivity and yield from raw cassava starch of 0.5 g-ethanol l(-1) x h(-1) and 0.45 g ethanol/g starch, respectively. In order to increase ethanol productivity, it was necessary to increase the dissolved oxygen (DO) concentration in the riser column and decrease the DO concentration in the downcomer column. However, increasing the aeration rate resulted in increases in the DO concentration in both the riser and the downcomer columns. At high aeration rate, there was no significant difference in the DO concentration in the riser and downcomer columns. The aeration rate was therefore uncoupled from the liquid circulation by attaching a time-controlled valve in the upper connecting pipe. By optimizing the time and frequency of valve opening, and operation at high aeration rate, it was possible to maintain a very high DO concentration in the riser column and a low DO concentration in the downcomer column. Under these conditions, ethanol productivity increased by more than 100%, to 1.17 g l(-1) x h(-1).     

Effects of environmental conditions on xylose fermentation by recombinant Escherichia coli.

In batch fermentations, optimal conversion of xylose to ethanol by recombinant Escherichia coli was obtained under the following conditions: 30 to 37 degrees C, pH 6.4 to 6.8, 0.1 to 0.2 M potassium phosphate buffer, and xylose concentrations of 8% or less. A yield of 39.2 g of ethanol per liter (4.9% ethanol by volume) was observed with 80 g of xylose per liter, equivalent to 96% of the maximum theoretical yield. Maximal volumetric productivity was 0.7 g of ethanol per liter per h in batch fermentations and 30 g of ethanol per liter per h in concentrated cell suspensions (analogous to cell recycling).     

Effects of environmental conditions on xylose fermentation by recombinant Escherichia coli

In batch fermentations, optimal conversion of xylose to ethanol by recombinant Escherichia coli was obtained under the following conditions: 30 to 37 degrees C, pH 6.4 to 6.8, 0.1 to 0.2 M potassium phosphate buffer, and xylose concentrations of 8% or less. A yield of 39.2 g of ethanol per liter (4.9% ethanol by volume) was observed with 80 g of xylose per liter, equivalent to 96% of the maximum theoretical yield. Maximal volumetric productivity was 0.7 g of ethanol per liter per h in batch fermentations and 30 g of ethanol per liter per h in concentrated cell suspensions (analogous to cell recycling).     

High production of acetone-butanol-ethanol with high cell density culture by cell-recycling and bleeding

A continuous acetone-butanol-ethanol (ABE) production system with high cell density obtained by cell-recycling of Clostridium saccharoperbutylacetonicum N1-4 has been studied. In conventional continuous culture of ABE without cell-recycling, the cell concentration was below 5.2 g l(-1) and the maximum ABE productivity was only 1.85 g l(-1)h(-1) at a dilution rate of 0.20 h(-1). To obtain a high cell density at a faster rate, we concentrated the solventogenic cells of the broth 10 times by membrane filtration and were able to obtain approximately 20 g l(-1) of active cells after only 12h of cultivation. Continuous culture with cell-recycling was then started, and the cell concentration increased gradually through cultivation to a value greater than 100 g l(-1). The maximum ABE productivity of 11.0 gl(-1)h(-1) was obtained at a dilution rate of 0.85 h(-1). However, a cell concentration greater than 100 gl(-1) resulted in heavy bubbling and broth outflow, which made it impossible to carry out continuous culture. Therefore, to maintain a stable cell concentration, cell-bleeding was performed together with cell-recycling. At dilution rates of 0.11h(-1) and above for cell-bleeding, continuous culture with cell-recycling could be operated for more than 200 h without strain degeneration and the overall volumetric ABE productivity of 7.55 gl(-1)h(-1) was achieved at an ABE concentration of 8.58 gl(-1).     

Xylitol formation by Candida boidinii in oxygen limited chemostat culture

Summary Production of xylitol by Candida boidinii NRRL Y-17213 occurs under conditions of an oxygen limitation. The extent to which substrate is converted to xylitol and its coproducts (ethanol, other polyols, acetic acid), and the relative flow rates of substrate to energetic and biosynthetic pathways is controlled by the degree of oxygen limitation.With decrease in oxygen concentration in the inlet gas, for a constant dilution rate of 0.05 1/h. the specific oxygen uptake rate decreased from 1.30 to 0.36 mmol/gh Xylitol was not produced at specific oxygen uptake rates above 0.91 mmol/gh. Upon shift to lower oxygen rates, specific xylitol production rate increased more rapidly than specific ethanol production rate:Nomenclature D dilution rate (1/h) - DOT dissolved oxygen tension (%) - m_o2 maintenance coefficient (mmol O₂/g cell mass h) - q_o2 specific oxygen uptake rate (mmol O₂/g cell mass h) - q_s specific xylose uptake rate (g xylose/g cell mass h) or (mmol xylose/g cell mass h) - q_x specific xylitol production rate (g xylitol/ g cell mass h) or (mmol xylitol/ g cell mass h) - q_e specific ethanol production rate (g ethanol/ g cell mass h) or (mmol ethanol/ g cell mass h) - q_CO2 specific carbon dioxide production rate (mmol CO₂/g cell mass h) - S xylose concentration (g/1) - Y_cm/s cell mass yield coefficient, (g cell mass/mmol xylose) or (g cell mass/ g xylose consumed) - Y_cm/O2 cell mass yield coefficient, (g cell mass/mmol O₂) - Y_X/S xylitol yield coefficient (g xylitol/g xylose consumed) - Y_x/O2 xylitol yield coefficient (g xylitol/mmol O₂) - Y_e/s ethanol yield coefficient (g ethanol/g xylose consumed) - OUR oxygen uptake rate (mmol O₂/1h) - specific growth rate (1/h)     

Production of ethanol by Clostridium thermosaccharolyticum: I. Effect of cell recycle and environmental parameters

The direct microbial conversion (DMC) process for the production of ethanol from lignocellulosic biomass is limited by low volumetric ethanol production rates due to the low cell densities of Clostridium thermosaccharolyticum which is a key organism for ethanol production in this process. Hence, this study focuses on the use of a continuous- culture cell recycle system to improve the volumetric ethanol productivity and yield of the fermentation of xylose by C. thermosaccharolyticum. Early experiments with the continuous-culture cell recycle system showed a two-fold improvement in volumetric ethanol productivity. However, the ethanol yield at the higher dilution rates suffered because of the large amount of lactate produced. The manipulation of two environmental parameters-iron concentration in the nutrient medium and the N(2) purge rate of the fermentor headspace-allowed a dramatic reduction in the lactate production and a simultaneous improvement in the ethanol titer and yield. Under the improved conditions of increased iron concentration (12.5 mg/L FeSO(4) . 7H(2)O) and decreased N(2) purge rate (0.1 L/min), a continuous culture of C. thermosaccharolyticum operating at a dilution rate of 0.24 h(-1) and 50% cell recycle produced 8.6 g/L ethanol and less than 1 g/L each of acetate and lactate. The volumetric ethanol productivity was 2.2 g/L/h, which is 8 times larger than obtained for a continuous culture operated with no cell recycle and the same specific growth rate.     

Effects of NADH-preferring xylose reductase expression on ethanol production from xylose in xylose-metabolizing recombinant Saccharomyces cerevisiae

Efficient conversion of xylose to ethanol is an essential factor for commercialization of lignocellulosic ethanol. To minimize production of xylitol, a major by-product in xylose metabolism and concomitantly improve ethanol production, Saccharomyces cerevisiae D452-2 was engineered to overexpress NADH-preferable xylose reductase mutant (XR(MUT)) and NAD?-dependent xylitol dehydrogenase (XDH) from Pichia stipitis and endogenous xylulokinase (XK). In vitro enzyme assay confirmed the functional expression of XR(MUT), XDH and XK in recombinant S. cerevisiae strains. The change of wild type XR to XR(MUT) along with XK overexpression led to reduction of xylitol accumulation in microaerobic culture. More modulation of the xylose metabolism including overexpression of XR(MUT) and transaldolase, and disruption of the chromosomal ALD6 gene encoding aldehyde dehydrogenase (SX6(MUT)) improved the performance of ethanol production from xylose remarkably. Finally, oxygen-limited fermentation of S. cerevisiae SX6(MUT) resulted in 0.64 g l?1 h?1 xylose consumption rate, 0.25 g l?1 h?1 ethanol productivity and 39% ethanol yield based on the xylose consumed, which were 1.8, 4.2 and 2.2 times higher than the corresponding values of recombinant S. cerevisiae expressing XR(MUT), XDH and XK only.     

Effect of mixed organic substrate on α-tocopherol production by <Emphasis Type="Italic">Euglena gracilis</Emphasis> in photoheterotrophic culture

Effects of organic carbon sources on cell growth and alpha-tocopherol productivity in wild and chloroplast-deficient W14ZUL strains of Euglena gracilis under photoheterotrophic culture were investigated. In both strains, the increase in cell growth was particularly high when glucose was added as the sole organic carbon source. On the other hand, alpha-tocopherol production per dry cell weight was enhanced by adding ethanol. Ethanol addition also increased the chlorophyll concentration in wild strain and mitochondria activity in W14ZUL strain. For effective alpha-tocopherol production, the effects of mixture of glucose and ethanol were investigated. The results showed that, when a mixture of glucose (6 g/l) and ethanol (4 g/l) was used, alpha-tocopherol productivity per culture broth was 3.89 x 10(-2) mg l(-1) h(-1), which was higher than the value obtained without addition of organic carbon source (0.92 x 10(-2) mg l(-1) h(-1)). In addition, under fed-batch cultivation using an internally illuminated photobioreactor, the alpha-tocopherol production per culture broth was 23.43 mg/l, giving a productivity of 16.27 x 10(-2) mg l(-1) h(-1).     

Effect of air supplement on the performance of continuous ethanol fermentation system

For the purpose of improving ethanol productivity, the effect of air supplement on the performance of continuous ethanol fermentation system was studied. The effect of oxygen supplement on yeast concentration, cell yield, cell viability, extracellular ethanol concentration, ethanol yield, maintenance coefficient, specific rates of glucose assimilation, ethanol production, and ethanol productivity have been evaluated, using a high alcohol tolerant Saccharomyces cerevisiae STV89 strain and employing a continuous fermentor equipped with an accurate air metering system in the flow rate range 0-11 mL air/L/h. It was found that, when a small amount of oxygen up to about 80mu mol oxygen/L/h was supplied, the ethanol productivity was significantly enhanced as compared to the productivity of the culture without any air supplement. It was also found that the oxygen supplement improved cell viability considerably as well as the ethanol tolerance level of yeast. As the air supply rate was increased, from 0 to 11 mL air/L/h while maintaining a constant dilution rate at about 0.06 h(-1), the cell concentration increased from 2.3 to 8.2 g/L and the ethanol productivity increased from 1.7 to 4.1 g ethanol/L/h, although the specific ethanol production rate decreased slightly from 0.75 to 0.5 g ethanol/g cell/h. The ethanol yield was slightly improved also with an increase in air supply rate, from about 0.37 to 0.45 ethanol/g glucose. The maintenance coefficient increased by only a small amount with the air supplement. This kind of air supplement technique may very well prove to be of practical importance to a development of a highly productive ethanol fermentation process system especially as a combined system with a high density cell culture technique.     

Anaerobic xylose fermentation by recombinant Saccharomyces cerevisiae carrying XYL1, XYL2, and XKS1 in mineral medium chemostat cultures

For ethanol production from lignocellulose, the fermentation of xylose is an economic necessity. Saccharomyces cerevisiae has been metabolically engineered with a xylose-utilizing pathway. However, the high ethanol yield and productivity seen with glucose have not yet been achieved. To quantitatively analyze metabolic fluxes in recombinant S. cerevisiae during metabolism of xylose-glucose mixtures, we constructed a stable xylose-utilizing recombinant strain, TMB 3001. The XYL1 and XYL2 genes from Pichia stipitis, encoding xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively, and the endogenous XKS1 gene, encoding xylulokinase (XK), under control of the PGK1 promoter were integrated into the chromosomal HIS3 locus of S. cerevisiae CEN.PK 113-7A. The strain expressed XR, XDH, and XK activities of 0.4 to 0.5, 2.7 to 3.4, and 1.5 to 1.7 U/mg, respectively, and was stable for more than 40 generations in continuous fermentations. Anaerobic ethanol formation from xylose by recombinant S. cerevisiae was demonstrated for the first time. However, the strain grew on xylose only in the presence of oxygen. Ethanol yields of 0.45 to 0.50 mmol of C/mmol of C (0.35 to 0.38 g/g) and productivities of 9.7 to 13.2 mmol of C h(-1) g (dry weight) of cells(-1) (0.24 to 0.30 g h(-1) g [dry weight] of cells(-1)) were obtained from xylose-glucose mixtures in anaerobic chemostat cultures, with a dilution rate of 0.06 h(-1). The anaerobic ethanol yield on xylose was estimated at 0.27 mol of C/(mol of C of xylose) (0.21 g/g), assuming a constant ethanol yield on glucose. The xylose uptake rate increased with increasing xylose concentration in the feed, from 3.3 mmol of C h(-1) g (dry weight) of cells(-1) when the xylose-to-glucose ratio in the feed was 1:3 to 6.8 mmol of C h(-1) g (dry weight) of cells(-1) when the feed ratio was 3:1. With a feed content of 15 g of xylose/liter and 5 g of glucose/liter, the xylose flux was 2.2 times lower than the glucose flux, indicating that transport limits the xylose flux.     

High ethanol productivities using small Ca-alginate beads of immobilized cells of Zymomonas mobilis

Summary Zymomonas mobilis cells were immobilized into small 1 mm diameter beads of Ca-alginate in order to minimize mass transfer limitations and maximize immobilized cell activity. A combination of small bead size with a high cell concentration of 58 g dry wt. cell per lit. bead volume resulted in high ethanol productivities using a newly designed packed bed bioreactor system. Steady-state dilution rates ranging from 0.4 h^-1 to 3.9 h^-1 were run resulting in a maximum productivity of 102 g ethanol/l/h for an inlet substrate concentration of 100 g glu/l and 87% conversion. The bioreactor was run continuously at a fixed dilution rate for 384 h and short intermittent treatment of the beads with CaCl₂ temporarily increased ethanol productivity to a maximum of 116 g ethanol/l/h.     

Continuous ethanol production from pineapple cannery waste using immobilized yeast cells

The cells of Saccharomyces cerevisiae ATCC 24553, were immobilized in k-carrageenan and packed in a tapered glass column reactor for ethanol production from pineapple cannery waste at temperature 30 degrees C and pH 4.5. The maximum productivity was 42.8 g ethanol 1(-1) h(-1) at a dilution rate of 1.5 h(-1). The volumetric ethanol productivity of the immobilized cells was ca. 11.5 times higher than the free cells. The immobilized cell reactor was operated over a period of 87 days at a dilution rate of 1.0 h(-1), without any loss in the immobilized cell activity. The maximum specific ethanol productivity and specific sugar uptake rate of the immobilized cells were 1.2 g ethanol g(-1) dry wt. cell h(-1) and 2.6 g sugar g(-1) dry wt. cell h(-1), respectively, at a dilution rate of 1.5 h(-1).     

Production of a high concentration acetic acid by Acetobacter aceti using a repeated fed-batch culture with cell recycling

Summary The acetic acid concentration in a batch culture of Acetobacter aceti M23 increased up to 90 g/l by adding ethanol intermittently. Although the bacterial cells ceased growth at about 60 g acetic acid/l, non-viable cells still preserved ethanol oxidation activity. Cell recycling by filtration in a repeated fed-batch culture increased the overall acetic acid production rate 2.84-fold compared to that without cell recycling for the purpose of obtaining an acetic acid concentration of 80.8 g/l. Repeated fed-batch cultivation with cell recycle was effective for increasing the production rate of acetic acid and obtaining high amounts close to a lethal concentration (90 g/l).Offprint requests to: Kiyoshi Toda     

Ethanol production from wood hydrolysate using genetically engineered Zymomonas mobilis

An ethanologenic microorganism capable of fermenting all of the sugars released from lignocellulosic biomass through a saccharification process is essential for secondary bioethanol production. We therefore genetically engineered the ethanologenic bacterium Zymomonas mobilis such that it efficiently produced bioethanol from the hydrolysate of wood biomass containing glucose, mannose, and xylose as major sugar components. This was accomplished by introducing genes encoding mannose and xylose catabolic enzymes from Escherichia coli. Integration of E. coli manA into Z. mobilis chromosomal DNA conferred the ability to co-ferment mannose and glucose, producing 91 % of the theoretical yield of ethanol within 36 h. Then, by introducing a recombinant plasmid harboring the genes encoding E. coli xylA, xylB, tal, and tktA, we broadened the range of fermentable sugar substrates for Z. mobilis to include mannose and xylose as well as glucose. The resultant strain was able to ferment a mixture of 20 g/l glucose, 20 g/l mannose, and 20 g/l xylose as major sugar components of wood hydrolysate within 72 h, producing 89.8 % of the theoretical yield. The recombinant Z. mobilis also efficiently fermented actual acid hydrolysate prepared from cellulosic feedstock containing glucose, mannose, and xylose. Moreover, a reactor packed with the strain continuously produced ethanol from acid hydrolysate of wood biomass from coniferous trees for 10 days without accumulation of residual sugars. Ethanol productivity was at 10.27 g/l h at a dilution rate of 0.25 h(-1).     

Salt accumulation resulting from base added for pH control, and not ethanol, limits growth of Thermoanaerobacteriumthermosaccharolyticum HG-8 at elevated feed xylose concentrations in continuous culture

Thermoanaerobacter thermosaccharolyticum HG-8 was grown in continuous culture to characterize growth limitation at high feed substrate and product concentrations. Continuous fermentation of 50 and 73 g/L xylose at a dilution rate based on the feed flow, D(f), of 0.053 h(-)(1) and with the pH controlled at 7.0 by addition of KOH resulted in steady state utilization of >99% of the xylose fed and production of ethanol and acetic acid at a mass ratio of about 2:1. Continuous cultures of T. thermosaccharolyticum growing at D(f) = 0.053 h(-)(1) achieved complete utilization of 75 g/L xylose in the presence of 19.1 g/L K(+) (0.49 M) and an ethanol concentration of 22.4 g/L ethanol. When the feed to a culture initially at steady state with a 75 g/L xylose feed and D(f) = 0.053 h(-)(1) was increased to 87.5 g/L xylose, limitation of growth and xylose utilization was observed. This limitation was not relieved by repeating this feed upshift experiment in the presence of increased nutrient levels and was not reproduced by addition of ethanol to a steady-state culture fed with 75 g/L xylose. By contrast, addition of KCl to a steady-state culture fed with 75 g/L xylose reproduced the K(+) concentration, limitation of growth and xylose utilization, and product concentration profiles observed in the feed upshift experiment. The maximum concentration at which growth of batch cultures was observed was 0.43 M for KCl, NaCl, and equimolar mixtures of these salts, suggesting that the observed limitation is not ion-specific. These data support the interpretation that inhibition salt accumulation resulting from addition of KOH for pH control is the limiting factor manifested in the feed upshift experiment and that both nutrient limitation and ethanol inhibition played little or no role as limiting factors. More generally, salt inhibition would appear to be a possible explanation for the discrepancy between the tolerance to added ethanol and the maximum concentration of produced ethanol reported in the literature for fermentation studies involving thermophilic bacteria.     

The effect of pH,temperature and sucrose concentration on high productivity continuous ethanol fermentation using Zymomonas mobilis

Summary A flocculent strain of Zymomonas mobilis was used for ethanol production from sucrose. Using a fermentor with cell recycle (internal and external settler) high sugar conversion and ethanol productivity were obtained. At a dilution rate of 0.5 h^-1 (giving 96% sugar conversion) the ethanol productivity, yield and concentrations respectively were 20 g/l/h, 0.45 g/g and 40 g/l using a medium containing 100 g/l sucrose. At a sucrose concentration of 150 g/l, the ethanol concentration reached 60 g/l. The ethanol yield was 80% theoretical due to levan and fructo-oligomer formation. No sorbitol was detected. This fermentation was conducted at a range of conditions from 30 to 36°C and from pH 4.0 to 5.5.