Effect of carbon dioxide on yeast growth and fermentation

Little is known about how the general lack of efficiency with which recombinant Saccharomyces cerevisiae strains utilize xylose affects the yeast metabolome. Quantitative metabolomics was therefore performed for two xylose-fermenting S. cerevisiae strains, BP000 and BP10001, both engineered to produce xylose reductase (XR), NAD ؉ -dependent xylitol dehydrogenase and xylulose kinase, and the corresponding wild-type strain CEN.PK 113-7D, which is not able to metabolize xylose. Contrary to BP000 expressing an NADPH-preferring XR, BP10001 expresses an NADH-preferring XR. An updated protocol of liquid chromatography/tandem mass spectrometry that was validated by applying internal 13 C-labeled metabolite standards was used to quantitatively determine intracellular pools of metabolites from the central carbon, energy, and redox metabolism and of eight amino acids. Metabolomic responses to different substrates, glucose (growth) or xylose (no growth), were analyzed for each strain. In BP000 and BP10001, flux through glycolysis was similarly reduced (ϳ27-fold) when xylose instead of glucose was metabolized. As a consequence, (i) most glycolytic metabolites were dramatically (<120-fold) diluted and (ii) energy and anabolic reduction charges were affected due to decreased ATP/AMP ratios (3-to 4-fold) and reduced NADP ؉ levels (ϳ3-fold), respectively. Contrary to that in BP000, the catabolic reduction charge was not altered in BP10001. This was due mainly to different utilization of NADH by XRs in BP000 (44%) and BP10001 (97%). Thermodynamic analysis complemented by enzyme kinetic considerations suggested that activities of pentose phosphate pathway enzymes and the pool of fructose-6-phosphate are potential factors limiting xylose utilization. Coenzyme and ATP pools did not rate limit flux through xylose pathway enzymes.Most of the biomass-to-ethanol processes that are currently advancing to the commercial-production scale rely on microbial strains for efficient fermentation of all sugars, that is, hexoses (mainly D-glucose) and pentoses (D-xylose, L-arabinose), contained in hydrolysates of lignocellulosic feedstocks (15, 36,65). The yeast Saccharomyces cerevisiae is among the top candidate microorganisms to be used for mixed hexosepentose fermentation (15). However, because neither xylose nor L-arabinose is a substrate naturally utilized by S. cerevisiae, extensive metabolic redesign was required to obtain strains producing pentose-derived ethanol in a useful yield (16,21, 37). Despite notable achievements, even the current best-performing yeast strains fall short in specific ethanol productivity (q ethanol ; 0.13 to 0.45 g ethanol/g biomass/h) from xylose compared to the corresponding q ethanol from glucose (1.2 g/g biomass/h) (33, 50). The low q ethanol for pentose fermentation is the result of at least two limiting factors. First of all, the yield coefficient (Y ethanol/sugar ) for consumption of pentose substrates is usually far lower (0.30 to 0.46) than theoretically expected (Y ethanol/xylose ϭ 0.51), reflecting the ...

Section: Methodsmentioning

confidence: 99%

Limitations in Xylose-FermentingSaccharomyces cerevisiae, Made Evident through Comprehensive Metabolite Profiling and Thermodynamic Analysis

Klimacek

Krahulec

Sauer

et al. 2010

“…The toxicity of CO 2 has been demonstrated for a wide range of microorganisms (5,6,12,17). Carbon dioxide, while exerting antimicrobial activity, causes little harm in foods, additives, etc.…”

mentioning

confidence: 99%

Accelerated Death Kinetics ofAspergillus nigerSpores under High-Pressure Carbonation

Shimoda

Kago

Kojima

et al. 2002

The death kinetics of Aspergillus niger spores under high-pressure carbonation were investigated with respect to the concentration of dissolved CO 2 (dCO 2 ) and treatment temperature. All of the inactivation followed first-order death kinetics. The D value (decimal reduction time, or the time required for a 1-log-cycle reduction in the microbial population) in the saline carbonated at 10 MPa was 0.16 min at 52°C. The log D values were linearly related to the treatment temperature and the concentration of dCO 2 , but a significant interaction was observed between them.Heat treatment in food processing is a broadly effective method for inactivating human pathogens and spoilage microorganisms. However, the high temperature involved can cause undesirable changes in nutritional and sensory properties.The toxicity of CO 2 has been demonstrated for a wide range of microorganisms (5,6,12,17). Carbon dioxide, while exerting antimicrobial activity, causes little harm in foods, additives, etc.; therefore, it is a suitable agent for controlling food spoilage microorganisms (22,23,29). In recent years, the influence of high-pressure CO 2 on the vegetative cells of various species has been demonstrated (1,3,4,7,8,9,10,11,13,14,18,20,28).We succeeded in dissolving CO 2 in an aqueous medium to a nearly saturated level by supplying high-pressure CO 2 in microbubbles (high-pressure carbonation). As a consequence, the treatment time and temperature required for microbial inactivation could be substantially reduced (15,16,26). Recently, it was shown that the antimicrobial effect was not related to the pressure of CO 2 but to the concentration of dissolved CO 2 (dCO 2 ) (27). The experiments were carried out with saturated dCO 2 concentrations under various combinations of pressure and temperature conditions.Most spoilages of fruit juice are caused by yeast and mold, because the germination of bacterial spores is inhibited at the pH of fruit juice (30). Shimoda et al. (27) have reported that the inactivation temperature of Saccharomyces cerevisiae was reduced by about 30°C under high-pressure carbonation. As mold spores have only a moderate thermal resistance, the heat treatment in fruit juice production is intended primarily to inactivate mold spores. The effects of pressurized CO 2 on the inactivation of mold spores, however, have not been investigated. A mesophilic mold, Aspergillus niger, was used as a test strain because it is a common contaminant of foods and other products (24).The purpose of the present paper was to investigate separately the effects of dCO 2 concentration, treatment pressure, and temperature on the death kinetics of Aspergillus niger spores. Fungal strain. Spores of Aspergillus niger (ATCC 16888) were obtained from the Japan Collection of Microorganisms (Wako, Saitama, Japan).Preparation of the spore suspension of A. niger. The strain was cultured on potato dextrose agar plates (Eiken Chemical Co., Ltd., Tokyo, Japan) at 25°C over 10 days. Spores were collected by washing the surface of the culture. Final...

“…Although the observed differences were not statistically significant for the anaerobic cultures, the observed changes were consistent in replicate experiments (i.e., the lack of statistical significance is not the result of some positive and some negative differences), and they are therefore regarded as being trends worth noting. Based on the examination of this one enzyme which catalyzes a CO 2 -producing reaction, our results cannot necessarily be extrapolated to carboxylases and decarboxylases in general (22,25). The results can, however, possibly be linked to the observed changes in the gene expression and cell membrane as suggested at the end of this discussion.…”

Section: Discussionmentioning

confidence: 79%

“…The IDH enzyme was chosen for several reasons: (i) it catalyzes a CO 2 -producing reaction and is therefore assumed to be affected by CO 2 (22,25), (ii) it only catalyzes one reaction, (iii) the enzyme was known to exist in the organism (14,41), and (iv) an IDH kit was commercially available. The slight but significant increase in IDH activity in the presence of CO 2 and oxygen is in contrast to the findings in the gram-negative Pseudomonas aeruginosa (25) but in accordance with those in the yeast Sclerotium rolfsii (28).…”

Section: Discussionmentioning

confidence: 99%

CO ₂ - and Anaerobiosis-Induced Changes in Physiology and Gene Expression of Different Listeria monocytogenes Strains

Jydegaard-Axelsen

Høiby

Holmstrøm

et al. 2004

Although carbon dioxide (CO 2 ) is known to inhibit growth of most bacteria, very little is known about the cellular response. The food-borne pathogen Listeria monocytogenes is characterized by its ability to grow in high CO 2 concentrations at refrigeration temperatures. We examined the listerial responses of different strains to growth in air, 100% N 2 , and 100% CO 2 . The CO 2 -induced changes in membrane lipid fatty acid composition and expression of selected genes were strain dependent. The acid-tolerant L. monocytogenes LO28 responded in the same manner to CO 2 as to other anaerobic, slightly acidic environments (100% N 2 , pH 5.7). An increase in the expression of the genes encoding glutamate decarboxylase (essential for survival in strong acid) as well as an increased amount of branched-chain fatty acids in the membrane was observed in both atmospheres. In contrast, the acid-sensitive L. monocytogenes strain EGD responded differently to CO 2 and N 2 at the same pH. In a separate experiment with L. monocytogenes 412, an increased isocitrate dehydrogenase activity level was observed for cells grown in CO 2 -containing atmospheres. Together, our findings demonstrate that the CO 2 -response is a partly strain-dependent complex mechanism. The possible links between the CO 2 -dependent changes in isocitrate dehydrogenase activity, glutamate metabolism and branched fatty acid biosynthesis are discussed.Modified atmosphere packaging (MAP) of foods is used to improve shelf life and safety by inhibition of spoilage organisms and pathogens. The gases used in MAP are most often combinations of oxygen (O 2 ), nitrogen (N 2 ), and carbon dioxide (CO 2 ). In most cases, the bacteriostatic effect is obtained by a combination of decreased O 2 and increased CO 2 concentrations (12). CO 2 is colorless, odorless, noncombustible, and bacteriostatic (21). It is both water and lipid soluble (12), and it does not leave toxic residues on MAP-stored foods (19).In food preservation MAP is frequently used for fresh muscle foods and ready-to-use vegetables stored and displayed at chill temperatures (12). The solubility of CO 2 increases at low temperatures (29), and it has recently been demonstrated that bacterial growth inhibition is proportional to the concentration of CO 2 dissolved in the water phase of a food matrix (8). The solubility of CO 2 is increased at neutral pH, high water activity, high buffer capacity, and high headspace-to-sample ratio (29).Listeria monocytogenes is a food-borne pathogen that can cause the severe human infection listeriosis (2). Cases of listeriosis caused by the consumption of contaminated meat or meat products have been reported since the 1990s (13), and several national prevention programs have been initiated (15). The organism is able to grow in the presence of CO 2 at low temperatures (2). CO 2 concentrations above 70% (N 2 balance gas) are required to inhibit growth of L. monocytogenes (11,17,20,27,43) at chill temperatures (Ͻ7°C), but even at such high CO 2 concentrations, the presence of ju...