Xylitol production from barley bran hydrolysates by continuous fermentation with Debaryomyces hansenii

The continuous bioconversion of xylose-containing solutions (obtained by acid hydrolysis of barley bran) into xylitol was carried out using the yeast Debaryomyces hansenii under microaerophilic conditions with or without cell recycle. In fermentations without cell recycle, the volumetric productivities ranged from 0.11–0.6 g l^–1 h^–1 were obtained for dilution rates of 0.008–0.088 h^–1. In experiments performed with cell recycle after membrane separation, the optimum xylitol productivity (2.53 g l^–1 h^–1) was reached at a dilution rate of 0.284 h^–1.     

Rapid ethanol fermentations using vacuum and cell recycle

Cell recycle and vacuum fermentation systems were developed for continuous ethanol production. Cell recycle was employed in both atmospheric pressure and vacuum fermentations to achieve high cell densities and rapid ethanol fermentation rates. Studies were conducted with Saccharomyces cerevisiae (ATCC No. 4126) at a fermentation temperature of 35°C. Employing a 10% glucose feed, a cell density of 50 g dry wt/liter was obtained in atmospheric-cell recycle fermentations which produced a fermentor ethanol productivity of 29.0 g/liter-hr. The vacuum fermentor eliminated ethanol inhibition by boiling away ethanol from the fermenting beer as it was formed. This permitted the rapid and complete fermentation of concentrated sugar solutions. At a total pressure of 50 mmHg and using a 33.4% glucose feed, ethanol productivities of 82 and 40 g/liter-hr were achieved with the vacuum system with and without cell recycle, respectively. Fermentor ethanol productivities were thus increased as much as twelvefold over conventional continuous fermentations. In order to maintain a viable yeast culture in the vacuum fermentor, a bleed of fermented broth had to be continuously withdrawn to remove nonvolatile compounds. It was also necessary to sparge the vacuum fermentor with pure oxygen to satisfy the trace oxygen requirement of the fermenting yeast.     

Microbial enzyme production in a membrane bioreactor

Summary Erwinia chrysanthemi cells were used to study the possibility of producing bacterial enzymes in a bioreactor coupled with a membrane filtration unit. Continuous fermentations with total cell recycle failed to give good production of pectate lyase (PL). Enzymatic, mechanical and physico-chemical damages were involved in this phenomenon. With a sequential recycle mode, we obtained productivity of 1.5 units·h^–1·1^–1 with a high PL concentration. Protease accumulation occurred when the bioreactor was coupled to a filtration unit. Moreover we have observed no loss of activity due to high shear stress caused by pumping. Offprint requests to: P. Boyaval     

Evaluation of the use of malic acid decarboxylase‐deficient starter culture in NaCl‐free cucumber fermentations to reduce bloater incidence

Aims

Accumulation of carbon dioxide (CO₂) in cucumber fermentations is known to cause hollow cavities inside whole fruits or bloaters, conducive to economic losses for the pickling industry. This study focused on evaluating the use of a malic acid decarboxylase (MDC)‐deficient starter culture to minimize CO₂ production and the resulting bloater index in sodium chloride‐free cucumber fermentations brined with CaCl₂.

Methods and Results

Attempts to isolate autochthonous MDC‐deficient starter cultures from commercial fermentations, using the MD medium for screening, were unsuccessful. The utilization of allochthonous MDC‐deficient starter cultures resulted in incomplete utilization of sugars and delayed fermentations. Acidified fermentations were considered, to suppress the indigenous microbiota and favour proliferation of the allochthonous MDC‐deficient Lactobacillus plantarum starter cultures. Inoculation of acidified fermentations with L. plantarum alone or in combination with Lactobacillus brevis minimally improved the conversion of sugars. However, inoculation of the pure allochthonous MDC‐deficient starter culture to 10⁷ CFU per ml in acidified fermentations resulted in a reduced bloater index as compared to wild fermentations and those inoculated with the mixed starter culture.

Conclusions

Although use of an allochthonous MDC‐deficient starter culture reduces bloater index in acidified cucumber fermentations brined with CaCl₂, an incomplete conversion of sugars is observed.

Significance and Impact of the Study

Economical losses due to the incidence of bloaters in commercial cucumber fermentations brined with CaCl₂ may be reduced utilizing a starter culture to high cell density.     

Fermentation kinetics of Zymomonas mobilis near zero growth rate

The fermentation kinetics Zymomonas mobilis were studied near zero growth rate in fed-batch cultures and continuous cultures with complete cell recycle. The results show the ethanol enhances that specific substrate conversion rate under these conditions. The maximum achievable ethanol concentration in continuous cultures with cell recycle (66 g/L) was significantly lower than in fed-batch cultures (100 g/L). The results indicate that growth-rate-independent metabolism is not instantaneous and can lag behind steadily increasing ethanol concentrations in fed-batch fermentations. A model is proposed to account for this slow adaptation.     

Comparison between in vivo and in vitro enzyme activities in continuous and batch fermentations of Clostridium acetobutylicum

Summary In vitro activities of key enzymes and related parameters (ATP and ADP concentrations, intracellular pH (pH _i ), cell volume and the transmembrane pH) in various continuous and batch fermentations of Clostridium acetobutylicum were studied in order to investigate the regulation (genetic vs. enzyme level) of the solventogenesis process. In vitro activities varied significantly among an acidogenic (glucose limited) and three solventogenic (an iron limited, a CO gassed and a biomass recycle) continuous fermentations. However, in vitro enzyme activities did not correlate with in vivo specific production rates in continuous cultures indicating that solvent formation is regulated primarily at the enzyme level. Carbon monoxide (CO) gassing of an acidogenic continuous culture resulted in butyrate uptake without acetone formation due to inactivation of the acetoacetate decarboxylase by CO. In continuous, and to some extent in batch cultures, butyrate can be taken up via the reversal of the butyrate kinase and phosphotransbutyrylase pathway. Solvent formation in batch fermentations is both a result of enzyme induction and regulation. Acetone formation and the induction of acetoacetate decarboxylase occur simultaneously whereas both alcohol dehydrogenases are induced several hours before initiation of alcohol production. Finally, the levels of intracellular and related cell parameters (pH _i , pH, ATP and ADP concentrations) are discussed and related to the possible mechanisms of solventogenesis.     

Solid-state fermentation of natural birch lignin by Phanerochaete chrysosporium

A strain of white rot fungus, Phanerochaete chrysosporium Burds. ME446, has been characterized with respect to the extent and rate of Betula nigrificans lignin and non-lignin conversion by solid-substrate fermentation for different culture conditions. Moisture content, inoculum density, nitrogen supplementation and autoclaving of birch solids significantly affected lignin conversion rates and yields in 20 day fermentations. Oxygen favoured lignin over non-lignin conversion at partial pressures of 1.0 atm. Oxygen pressures of 2.0 atm severely inhibited both lignin and non-lignin conversions. Carbon dioxide partial pressures of 0.25, 0.5 and 1.0 atm at oxygen pressures of 1.0 atm increasingly inhibited both lignin and non-lignin conversion rates and yields. The results of these studies demonstrate the effects of major process variables and suggest a need to control the gas environment for process optimization.     

Advances in biotechnological production of butyric acid

The review is focused on several aspects of butyric acid production: butyric acid-producing bacterial strains, the characteristics of the genus Clostridium (the bacterium most used for butyrate production), and alternative methods of obtaining butyric acid by alcohol biotransformation. Further, the main metabolic pathways of butyrate production, and possibilities for their control are outlined. Batch, fed-batch or continuous fermentation combined with cell recycle or immobilization are applicable for anaerobic fermentations using Clostridium as the production strain. The best process comprises a combination of high cell concentration and slowly growing biomass, in addition to high production selectivity and low inhibitory effects of the end-product. Inhibitory effects may be avoided by on-line removal of the end-product. Extraction alone or extraction combined with simultaneous stripping of the organic phase (liquid membrane) into the second aqueous phase (pertraction) seem to be the most suitable methods for on-line butyrate removal. The biocompatibility and the distribution coefficient of the organic phase under fermentation conditions should be considered before designing a fermentation apparatus. Journal of Industrial Microbiology & Biotechnology (2000) 24, 153–160. Received 12 August 1999/ Accepted in revised form 03 December 1999     

Oxygen starvation induces cell death in Candida shehatae fermentations of d-xylose, but not d-glucose

Candida shehatae cells, cultivated on d-glucose and d-xylose, were subjected to a shift from fully aerobic to anaerobic fermentative conditions. After anaerobic conditions were imposed, growth was limited to approximately one doubling or less as C. shehatae rapidly entered a stationary phase of growth. Following the shift to anoxia, cell viability rapidly declined and the total cell volume declined in the d-xylose fermentations. Moreover, the cell volume distribution shifted to smaller volumes. Cell viability, measured by plate counts, declined nine times faster for d-xylose fermentations than for d-glucose fermentations. Anaerobic growth did not occur on either d-glucose or d-xylose. Selected vitamins and amino acids did not stimulate anaerobic growth in C. shehatae, but did enhance anaerobic growth on d-glucose in S. cerevisiae. The decline in cell viability and lack of anaerobic growth by C. shehatae were attributed to oxygen deficiency and not to ethanol inhibition. The results shed light on why C. shehatae anaerobic fermentations are not currently practical and suggest that research directed towards a biochemical understanding of why C. shehatae can not grow anaerobically will yield significant improvements in ethanol fermentations from d-xylose. Received 26 October 1998 / Received revision: 26 January 1999 / Accepted: 12 February 1999     

Production of acetone-butanol by extractive fermentation using dibutylphthalate as extractant

Extractive fermentation of glucose, glucose-xylose mixtures and hydrolysates of lignocellulosics to acetone-butanol solvents were studied and compared with similar fermentations in the absence of extractant. The extractant selected for this research was dibutylphthalate which, in addition to having satisfactory physical properties for this purpose, is non-toxic and mildly stimulating to the growth of the organism used, Clostridium acetobutylicum P262. Sugar concentrations mainly in the range of 80 to 100 g/l resulted in solvent concentrations of 28 to 30 g/l in 24 h extractive fermentations, compared to 18 to 20 g/l for non-extractive control fermentations. Conversion factors of 0.33 to 0.37 g solvents/g sugar consumed were obtained. Rapid fermentation was achieved by high cell concentrations and cell recycle from each 24 h fermentation to the succeeding similar 24 h fermentation. Somewhat higher nutrients were also helpful. By this means, 255 l of acetone-butanol solvents were obtained per tonne of aspen wood, 298 l per tonne of pine and 283 l per tonne of corn stover. Such high product yields from inexpensive substrates offer the prospect of economic viability for the process.     

Effect of xylitol on the metabolism and viability of Candida shehatae

Anaerobic D-xylose fermentations were performed with C. shehatate in the presence of 0, 25, and 50 g/L of xylitol. D-Xylose was preferentially utilized over xylitol and ethanol yields (Y _Etoh/S 0.26 g/g) were unaffected by xylitol. Added xylitol did inhibit conversion of xylose to xylitol at an external xylitol concentration of 50 g/L; Y _Xylitol/S was reduced from 0.21 to 0.14. Cell viability declined in all of the fermentations, but was not due to the presence of xylitol. The decline in viability was attributed to oxygen deprivation, since ethanol levels only reached 10.5 g/L and the decline cell viability was the same in each fermentation, regardless of the xylitol concentration.     

The role of oxygen in yeast metabolism during high cell density brewery fermentations

The volumetric productivity of the beer fermentation process can be increased by using a higher pitching rate (i.e., higher inoculum size). However, the decreased yeast net growth observed in these high cell density fermentations can have a negative impact on the physiological stability throughout subsequent yeast generations. The use of different oxygen conditions (wort aeration, wort oxygenation, yeast preoxygenation) was investigated to improve the growth yield during high cell density fermentations and yeast metabolic and physiological parameters were assessed systematically. Together with a higher extent of growth (dependent on the applied oxygen conditions), the fermentation power and the formation of unsaturated fatty acids were also affected. Wort oxygenation had a significant decreasing effect on the formation of esters, which was caused by a decreased expression of the alcohol acetyl transferase gene ATF1, compared with the other conditions. Lower glycogen and trehalose levels at the end of fermentation were observed in case of the high cell density fermentations with oxygenated wort and the reference fermentation. The expression levels of BAP2 (encoding the branched chain amino acid permease), ERG1 (encoding squalene epoxidase), and the stress responsive gene HSP12 were predominantly influenced by the high cell concentrations, while OLE1 (encoding the fatty acid desaturase) and the oxidative stress responsive genes SOD1 and CTT1 were mainly affected by the oxygen availability per cell. These results demonstrate that optimisation of high cell density fermentations could be achieved by improving the oxygen conditions, without drastically affecting the physiological condition of the yeast and beer quality.     

Inhibition of yeast by lactic acid bacteria in continuous culture: nutrient depletion and/or acid toxicity?

Lactic acid was added to batch very high gravity (VHG) fermentations and to continuous VHG fermentations equilibrated to steady state with Saccharomyces cerevisiae. A 53% reduction in colony-forming units (CFU) ml^–1 of S. cerevisiae was observed in continuous fermentation at an undissociated lactic acid concentration of 3.44% w/v; and greater than 99.9% reduction was evident at 5.35% w/v lactic acid. The differences in yeast cell number in these fermentations were not due to pH, since batch fermentations over a pH range of 2.5–5.0 did not lead to changes in growth rate. Similar fermentations performed in batch showed that growth inhibition with added lactic acid was nearly identical. This indicates that the apparent high resistance of S. cerevisiae to lactic acid in continuous VHG fermentations is not a function of culture mode. Although the total amount of ethanol decreased from 48.7 g l^–1 to 14.5 g l^–1 when 4.74% w/v undissociated lactic acid was added, the specific ethanol productivity increased ca. 3.2-fold (from 7.42×10^–7 g to 24.0×10^–7 g ethanol CFU^–1 h^–1), which indicated that lactic acid stress improved the ethanol production of each surviving cell. In multistage continuous fermentations, lactic acid was not responsible for the 83% (CFU ml^–1) reduction in viable S. cerevisiae yeasts when Lactobacillus paracasei was introduced to the system at a controlled pH of 6.0. The competition for trace nutrients in those fermentations and not lactic acid produced by L. paracasei likely caused the yeast inhibition.     

Impact of recycling stillage on conversion of dilute sulfuric acid pretreated corn stover to ethanol

Both the current corn starch to ethanol industry and the emerging lignocellulosic biofuels industry view recycling of spent fermentation broth or stillage as a method to reduce fresh water use. The objective of this study was to understand the impact of recycling stillage on conversion of corn stover to ethanol. Sugars in a dilute‐acid pretreated corn stover hydrolysate were fermented to ethanol by the glucose–xylose fermenting bacteria Zymomonas mobilis 8b. Three serial fermentations were performed at two different initial sugar concentrations using either 10% or 25% of the stillage as makeup water for the next fermentation in the series. Serial fermentations were performed to achieve near steady state concentration of inhibitors and other compounds in the corn stover hydrolysate. Little impact on ethanol yields was seen at sugar concentrations equivalent to pretreated corn stover slurry at 15% (w/w) with 10% recycle of the stillage. However, ethanol yields became progressively poorer as the sugar concentration increased and fraction of the stillage recycled increased. At an equivalent corn stover slurry concentration of 20% with 25% recycled stillage the ethanol yield was only 5%. For this microorganism with dilute‐acid pretreated corn stover, recycling a large fraction of the stillage had a significant negative impact on fermentation performance. Although this finding is of concern for biochemical‐based lignocellulose conversion processes, other microorganism/pretreatment technology combinations will likely perform differently. Biotechnol. Bioeng. 2010;105: 992–996. © 2009 Wiley Periodicals, Inc.     

Recent Developments in the Zymomonas Process for Ethanol Production

Abstract

The bacterium Zymomonas mobilis, which is used in the tropics to make pulque and alcoholic palm wines, appears to have considerable potential for industrial alcohol fermentations. Some of the advantages of the Zymomonas process reported in studies from our laboratory^1-24 are

1. There are significantly higher specific rates of sugar uptake and ethanol production compared to those found for yeasts.

2. Considerably higher volumetric ethanol productivities found in continuous cell recycle systems (up to 120 to 200 g/hr).

3. There are higher ethanol yields and lower biomass production than for yeasts. The lower biomass concentrations would seem to be a consequence of the lower metabolic energy available for growth. Zymomonas metabolize glucose via the Entner-Doudoroff pathway while yeasts convert glucose to ethanol via glycolysis.

4. Zymomonas cultures grow anaerobically and, unlike yeasts, do not require the controlled addition of oxygen to maintain viability at high cell concentrations.

5. The ethanol tolerance of some selected strains of Zymomonas is comparable if not higher than strains of Saccharomyces cerevisiae. Ethanol concentrations of 85 g/(up to 11% v/v) have been achieved in continuous culture and up to 130 g/(16% v/v) in batch culture.     

The ability to use nitrate confers advantage to <Emphasis Type="Italic">Dekkera bruxellensis</Emphasis> over <Emphasis Type="Italic">S. cerevisiae</Emphasis> and can explain its adaptation to industrial fermentation processes

The yeast Dekkera bruxellensis has been regarded as a contamination problem in industrial ethanol production because it can replace the originally inoculated Saccharomyces cerevisiae strains. The present study deals with the influence of nitrate on the relative competitiveness of D. bruxellensis and S. cerevisiae in sugar cane ethanol fermentations. The industrial strain D. bruxellensis GDB 248 showed higher growth rates than S. cerevisiae JP1 strain in mixed ammonia/nitrate media, and nitrate assimilation genes were only slightly repressed by ammonia. These characteristics rendered D. bruxellensis cells with an ability to overcome S. cerevisiae populations in both synthetic medium and in sugar cane juice. The results were corroborated by data from industrial fermentations that showed a correlation between high nitrate concentrations and high D. bruxellensis cell counts. Moreover, the presence of nitrate increased fermentation efficiency of D. bruxellensis cells in anaerobic conditions, which may explain the maintenance of ethanol production in the presence of D. bruxellensis in industrial processes. The presence of high levels of nitrate in sugar cane juice may be due to its inefficient conversion by plant metabolism in certain soil types and could explain the periodical episodes of D. bruxellensis colonization of Brazilian ethanol plants.     

Increased xylitol production rate during long-term cell recycle fermentation of Candida tropicalis

Long-term cell recycle fermentations of Candida tropicalis were performed over 14 rounds of fermentation. The average xylitol concentrations, fermentation times, volumetric productivities and product yields for 14 rounds were 105 g l^–1, 333 h, 4.4 g l^–1 h^–1 and 78%, respectively, in complex medium; and 110 g l^–1, 284 h, 5.4 g l^–1 h^–1 and 81%, respectively, in a chemically defined medium. These productivities were 1.7 and 2.4 times those with batch fermentation in the complex and chemically defined media, respectively. The xylitol yield from xylose with cell recycle fermentation using the chemically defined medium was 81% (w/w), which was 7% greater than the xylitol yield with batch fermentation (74%); both modes of fermentation gave the same yield using the complex medium. These results suggest that the chemically defined medium is more suitable for production of xylitol than complex medium.     

Computation of the fraction of induced cells in enzyme induction systems

A theoretical model is developed for continuous multistage enzyme production systems, which consist of a growth fermentor used for growing microorganisms rapidly without enzyme production and a subsequent system of induction reactors in which enzymes induction and production occurs. The model allows the computation of the fraction of induced cells residing in the induction reactor for organisms exhibiting a lag phase in enzyme induction. For this model a general analytical solution was obtained for the cumulative internal residence time distribution of a series of n well-stirred vessels with a recycle. The theoretical results are compared in a preliminary way with experimentally measured cellulase productivities of continuous multistage cellulose fermentations with Trichoderma viride QM 9414.     

Catalytic and biocatalytic oxidation of glucose to gluconic acid in a modified three-phase reactor

A comparative study of catalytic and biocatalytic glucose oxidation was carried out. Gluconobacter oxydans NBIMCC 1043 strain was used for biocatalytic glucose conversion. In the case of cell recycle coupled with cross-flow microfiltration the productivity and biomass concentration reached 40% and 3 g l^–1 respectively, in comparison to those of batch fermentation (21% and 2.3 g l^–1, respectively).     

Production of single cell protein from ethanol by fed-batch process

The production of single cell protein (SCP) form ethanol is an interesting process to study from a biochemical engineering viewpoint. The cellular yield mainly depends upon the metabolic activity of the cells and the amount of substrate available. Fedbatch fermentations Were run in a 70 liter highly instrumented computer-coupled fermentor using Candida utilis. Respiratory quotient and culture fluorescence, measuring NADH, indicate by which pathway ethanol is utilized. By monitoring these parameters it is possible to control the ethanol concentration so that accumulation of acetate is minimized and the conversion of ethanol to cell mass is maximized.