Formate as an Auxiliary Substrate for Glucose-Limited Cultivation of Penicillium chrysogenum: Impact on Penicillin G Production and Biomass Yield

Production of β-lactams by the filamentous fungus Penicillium chrysogenum requires a substantial input of ATP. During glucose-limited growth, this ATP is derived from glucose dissimilation, which reduces the product yield on glucose. The present study has investigated whether penicillin G yields on glucose can be enhanced by cofeeding of an auxiliary substrate that acts as an energy source but not as a carbon substrate. As a model system, a high-producing industrial strain of P. chrysogenum was grown in chemostat cultures on mixed substrates containing different molar ratios of formate and glucose. Up to a formate-to-glucose ratio of 4.5 mol·mol⁻¹, an increasing rate of formate oxidation via a cytosolic NAD⁺-dependent formate dehydrogenase increasingly replaced the dissimilatory flow of glucose. This resulted in increased biomass yields on glucose. Since at these formate-to-glucose ratios the specific penicillin G production rate remained constant, the volumetric productivity increased. Metabolic modeling studies indicated that formate transport in P. chrysogenum does not require an input of free energy. At formate-to-glucose ratios above 4.5 mol·mol⁻¹, the residual formate concentrations in the cultures increased, probably due to kinetic constraints in the formate-oxidizing system. The accumulation of formate coincided with a loss of the coupling between formate oxidation and the production of biomass and penicillin G. These results demonstrate that, in principle, mixed-substrate feeding can be used to increase the yield on a carbon source of assimilatory products such as β-lactams.     

Chemostat production of pediocin SM‐1 by Pediococcus pentosaceus Mees 1934

Production of the bacteriocin pediocin SM‐1 by Pediococcus pentosaceus Mees 1934 was investigated in pH‐controlled batch and chemostat cultures using a complex medium containing glucose, sucrose or fructose. In chemostat cultures operated at 150 rpm, 30°C, 60% dissolved oxygen tension, pH 6.5, and D = 0.148 h^?1, the pediocin titer reached 185 AU/mL representing an increase of 32% compared with batch cultures in which glucose was used as the carbon source. Pediocin biosynthesis was markedly affected by the growth rate of the producer microorganism. For all carbon sources tested, pediocin production appeared to take place only at dilution rates lower than μ_max. However, only glucose supported production at the very low dilution rate of 0.05 h^?1 indicating a direct regulation of pediocin biosynthesis by the carbon source. Glucose supported higher biomass productivity and higher pediocin titers and yields compared with the other sugars used. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1481–1486, 2015     

Effect of dilution rate and methanol‐glycerol mixed feeding on heterologous Rhizopus oryzae lipase production with Pichia pastoris Mut+ phenotype in continuous culture

The induction using substrate mixtures is an operational strategy for improving the productivity of heterologous protein production with Pichia pastoris. Glycerol as a cosubstrate allows for growth at a higher specific growth rate, but also has been reported to be repressor of the expression from the AOX1 promoter. Thus, further insights about the effects of glycerol are required for designing the induction stage with mixed substrates. The production of Rhizopus oryzae lipase (ROL) was used as a model system to investigate the application of methanol‐glycerol feeding mixtures in fast metabolizing methanol phenotype. Cultures were performed in a simple chemostat system and the response surface methodology was used for the evaluation of both dilution rate and methanol‐glycerol feeding composition as experimental factors. Our results indicate that productivity and yield of ROL are strongly affected by dilution rate, with no interaction effect between the involved factors. Productivity showed the highest value around 0.04–0.06 h^?1, while ROL yield decreased along the whole dilution rate range evaluated (0.03–0.1 h^?1). Compared to production level achieved with methanol‐only feeding, the highest specific productivity was similar in mixed feeding (0.9 UA g‐biomass^?1 h^?1), but volumetric productivity was 70% higher. Kinetic analysis showed that these results are explained by the effects of dilution rate on specific methanol uptake rate, instead of a repressor effect caused by glycerol feeding. It is concluded that despite the effect of dilution rate on ROL yield, mixed feeding strategy is a proper process option to be applied to P. pastoris Mut⁺ phenotype for heterologous protein production. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:707–714, 2015     

Fast dynamic response of the fermentative metabolism of Escherichia coli to aerobic and anaerobic glucose pulses

The response of Escherichia coli cells to transient exposure (step increase) in substrate concentration and anaerobiosis leading to mixed‐acid fermentation metabolism was studied in a two‐compartment bioreactor system consisting of a stirred tank reactor (STR) connected to a mini‐plug‐flow reactor (PFR: BioScope, 3.5 mL volume). Such a system can mimic the situation often encountered in large‐scale, fed‐batch bioreactors. The STR represented the zones of a large‐scale bioreactor that are far from the point of substrate addition and that can be considered as glucose limited, whereas the PFR simulated the region close to the point of substrate addition, where glucose concentration is much higher than in the rest of the bioreactor. In addition, oxygen‐poor and glucose‐rich regions can occur in large‐scale bioreactors. The response of E. coli to these large‐scale conditions was simulated by continuously pumping E. coli cells from a well stirred, glucose limited, aerated chemostat (D = 0.1 h^?1) into the mini‐PFR. A glucose pulse was added at the entrance of the PFR. In the PFR, a total of 11 samples were taken in a time frame of 92 s. In one case aerobicity in the PFR was maintained in order to evaluate the effects of glucose overflow independently of oxygen limitation. Accumulation of acetate and formate was detected after E. coli cells had been exposed for only 2 s to the glucose‐rich (aerobic) region in the PFR. In the other case, the glucose pulse was also combined with anaerobiosis in the PFR. Glucose overflow combined with anaerobiosis caused the accumulation of formate, acetate, lactate, ethanol, and succinate, which were also detected as soon as 2 s after of exposure of E. coli cells to the glucose and O₂ gradients. This approach (STR‐mini‐PFR) is useful for a better understanding of the fast dynamic phenomena occurring in large‐scale bioreactors and for the design of modified strains with an improved behavior under large‐scale conditions. Biotechnol. Bioeng. 2009; 104: 1153–1161. © 2009 Wiley Periodicals, Inc.     

A physiological and enzymatic study of Debaryomyces hansenii growth on xylose- and oxygen-limited chemostats

The effect of changing growth rate and oxygen transfer rate (OTR) on Debaryomyces hansenii physiology was studied using xylose-limited and oxygen-limited chemostat cultures, respectively, and complemented with enzymatic assays. Under xylose-limited chemostat (oxygen-excess), neither ethanol nor xylitol was produced over the entire range of dilution rate (D). The maximal volumetric biomass productivity was 2.5 g l^–1 h^–1 at D =0.25 h^–1 and cell yield was constant at all values of D. The respiratory rates and xylose consumption rate increased linearly with growth rate but, above 0.17 h^–1, oxygen consumption rate had a steeper increase compared to carbon dioxide production rate. Enzymatic analysis of xylose metabolism suggests that internal fluxes are redirected as a function of growth rate. For values of D up to 0.17 h^–1, the xylose reductase (XR) titre is lower than the xylitol dehydrogenase (XDH) titre, whereas above 0.17 h^–1 XR activity is about twice that of XDH and the NADPH-producing enzymes sharply increase their titres indicating an internal metabolic flux shift to meet higher NADPH metabolic requirements. Moreover, the enzymes around the pyruvate node also exhibited different patterns if D was above or below 0.17 h^–1. Under oxygen-limited chemostat (xylose-excess) the metabolism changed drastically and, due to oxidative phosphorylation limitation, cell yield decreased to 0.16 g g^–1 for an OTR of 1.4 mmol l^–1 h^–1 and xylitol became the major extracellular product along with minor amounts of glycerol. The enzymatic analysis revealed that isocitrate dehydrogenase is not regulated by oxygen, whereas XR, XDH and the NADPH-producing enzymes changed their levels according to oxygen availability. Electronic Publication     

Dilution rates influence ammonia-assimilating enzyme activities and cell parameters of Selenomonas ruminantium strain D in continuous (glucose-limited) culture

Aims: The objective of this study was to examine the effect of dilution rates (Ds, varying from 0·05 to 0·42 h^?1) in glucose‐limited continuous culture on cell yield, cell composition, fermentation pattern and ammonia assimilation enzymes of Selenomonas ruminantium strain D. Methods and Results: All glucose‐limited continuous culture experiments were conducted under anaerobic conditions. Except for protein, all cell constituents including carbohydrates, RNA and DNA yielded significant cubic responses to Ds with the highest values at Ds of either 0·10 or 0·20 h^?1. At Ds higher than 0·2 h^?1, fermentation acid pattern shifted primarily from propionate and acetate to lactate production. Succinate also accumulated at the higher Ds (0·30 and 0·42 h^?1). Glucose was most efficiently utilized by S. ruminantium D at 0·20 h^?1 after which decreases in glucose and ATP yields were observed. Under energy limiting conditions, glutamine synthetase (GS) and glutamate dehydrogenase (GDH) appeared to be the major enzymes involved in nitrogen assimilation suggesting that other potential ammonia incorporating enzymes were of little importance in ammonia assimilation in S. ruminantium D. GS exhibited lower activities than GDH at all Ds, which indicates that the bacterial growth rate is not a primary regulator of their activities. Conclusions: Studied dilution rates influenced cell composition, fermentation pattern and nitrogen assimilation of S. ruminantium strain D grown in glucose‐limited continuous culture. Significance and Impact of the Study: Selenomonas ruminantium D is an ecologically and evolutionary important bacterium in ruminants and is present under most rumen dietary conditions. Characterizing the growth physiology and ammonia assimilation enzymes of S. ruminantium D during glucose limitation at Ds, which simulate the liquid turnover rates in rumen, will provide a better understanding of how this micro‐organism responds to differing growth conditions.     

The effect of mixtures of glucose and formate on the yield and respiration of a chemostat culture of Beneckea natriegens

Beneckea natriegens oxidizes sodium formate constitutively when grown on glucose or glycerol in chemostat culture, but cannot utilize formate as the sole source of carbon and energy for growth. However, when grown on a mixture of glucose and formate (D=0.37 h^-1, pH 7.6) the yield is higher than on glucose alone.The yield, expressed in terms of g bacterial dry weight g^-1 glucose plus formate carbon utilized, gave a linear relationship when plotted against the total heat of combustion of glucose plus formate utilized. Extrapolation of the plot cut the abscissa at a value equivalent to the heat of combustion of formate, which suggests that formate is not utilised as a source of carbon but only energy.In cultures with nitrate as the sole source of nitrogen the yield from glucose was lower than that observed with ammonia but the addition of formate to the culture utilizing nitrate resulted in an increase in the yield from glucose to a value similar to that observed with ammonia.At a culture pH value of 7.65 unused formate (<0.15–227 mM) in the culture supernatant had no effect on respiration spiration or yield, but at a culture pH of 6.7 excess formate caused a marked increase in respiration rate and a large decrease in the yield from glucose; further decrease in the pH value caused washout of the culture. This may be explained by undissociated formic acid causing uncoupling of oxidative phosphorylation.     

Xylitol Production from D‐Xylose at Different Oxygen Transfer Coefficients in a Batch Bioreactor

Conversion of D‐xylose to xylitol by Candida boidinii NRRL Y‐17213 was studied under anaerobic and oxygen limited conditions by varying the oxygen transfer coefficient k_La. Shake flask experiments were used to provide the preliminary information required to perform experiments in a bioreactor. The yeast did not grow under fully anaerobic conditions, but anaerobic formations of xylitol, ethanol, ribitol, and glycerol were observed as well as D‐xylose assimilation of 11 %. In shake flasks, with an initial D‐xylose concentration of 50 g/L, an increase in k_La from 8 to 46 h^–1 resulted in a faster growth, higher rate of substrate uptake and lower yields of products. The highest xylitol productivity (0.052 g/L h) was attained at k_La = 8 h^–1. At k_La = 46 h^–1, 98.6 % of D‐xylose was consumed and mainly converted to biomass. Using 130 g/L D‐xylose, k_La was varied in the fermenter from 26 to 78 h^–1. The percentage of consumed D‐xylose increased from 31 % at k_La = 26 h^–1 to 93–94 % at all other aeration levels. Biomass yield increased with k_La, whereas ethanol, ribitol, and glycerol yields exhibited an opposite dependence on the oxygenation level. The most favorable oxygen transfer coefficient for xylitol formation, in the fermenter, was k_La = 47 h^–1 when its concentration (57.5 g/L) surpassed ethanol accumulation by 3.6‐fold, and the glycerol plus ribitol by 10‐fold. Concurrently, xylitol yield and productivity reached 0.45 g/g and 0.26 g/L h, respectively. The volumetric xylitol productivity was affected more by changes in the aeration than the corresponding yield.     

Acetone–butanol–ethanol production with high productivity using Clostridium acetobutylicum BKM19

Conventional acetone–butanol–ethanol (ABE) fermentation is severely limited by low solvent titer and productivities. Thus, this study aims at developing an improved Clostridium acetobutylicum strain possessing enhanced ABE production capability followed by process optimization for high ABE productivity. Random mutagenesis of C. acetobutylicum PJC4BK was performed by screening cells on fluoroacetate plates to isolate a mutant strain, BKM19, which exhibited the total solvent production capability 30.5% higher than the parent strain. The BKM19 produced 32.5 g L^?1 of ABE (17.6 g L^?1 butanol, 10.5 g L^?1 ethanol, and 4.4 g L^?1 acetone) from 85.2 g L^?1 glucose in batch fermentation. A high cell density continuous ABE fermentation of the BKM19 in membrane cell‐recycle bioreactor was studied and optimized for improved solvent volumetric productivity. Different dilution rates were examined to find the optimal condition giving highest butanol and ABE productivities. The maximum butanol and ABE productivities of 9.6 and 20.0 g L^?1 h^?1, respectively, could be achieved at the dilution rate of 0.85 h^?1. Further cell recycling experiments were carried out with controlled cell‐bleeding at two different bleeding rates. The maximum solvent productivities were obtained when the fermenter was operated at a dilution rate of 0.86 h^?1 with the bleeding rate of 0.04 h^?1. Under the optimal operational condition, butanol and ABE could be produced with the volumetric productivities of 10.7 and 21.1 g L^?1 h^?1, and the yields of 0.17 and 0.34 g g^?1, respectively. The obtained butanol and ABE volumetric productivities are the highest reported productivities obtained from all known‐processes. Biotechnol. Bioeng. 2013; 110: 1646–1653. © 2013 Wiley Periodicals, Inc.     

NEUTRAL LIPID AND CARBOHYDRATE PRODUCTIVITIES AS A RESPONSE TO NITROGEN STATUS IN ISOCHRYSIS SP. (T‐ISO; HAPTOPHYCEAE): STARVATION VERSUS LIMITATION1

Partitioning of the carbon (C) fixed during photosynthesis between neutral lipids (NL) and carbohydrates was investigated in Isochrysis sp. (Haptophyceae) in relation to its nitrogen (N) status. Using batch and nitrate‐limited continuous cultures, we studied the response of these energy reserve pools to both conditions of N starvation and limitation. During N starvation, NL and carbohydrate quotas increased but their specific growth rates (specific rates of variation, μ_CAR and μ_NL) decreased. When cells were successively deprived and then resupplied with NO₃, both carbohydrates and neutral lipids were inversely related to the N quota (N:C). These negative relationships were not identical during N impoverishment and replenishment, indicating a hysteresis phenomenon between N and C reserve mobilizations. Cells acclimated to increasing degrees of N limitation in steady‐state chemostat cultures showed decreasing NL quota and increasing carbohydrate quota. N starvation led to a visible but only transient increase of NL productivity. In continuous cultures, the highest NL productivity was obtained for the highest experimented dilution rate (D = 1.0 d^?1; i.e., for non N‐limited growth conditions), whereas the highest carbohydrate productivity was obtained at D = 0.67 d^?1. We used these results to discuss the nitrogen conditions that optimize NL productivities in the context of biofuel production.     

Legume-Rhizobium interactions: host induced alterations in capsular polysaccharides and infectivity of cowpea Rhizobia

Klebsiella aerogenes NCTC418 was cultured anaerobically under glucose-limited conditions in chemostat cultures at various growth rates, ranging from 0.13 h^-1 to 0.82 h^-1. It was found that the specific uptake rate of glucose varied linearly with the growth rate and that under these conditions glucose was fermented solely to acetate and ethanol plus CO₂+H₂ and formate.When steady-state cultures were pulsed with cell saturating concentrations of glucose, the specific glucose aptake rate increased immediately and substantially. However, at steady-state growth rates lower than 0.5 h^-1, this increase was not accompanied by a change in the growth rate, in contrast to cultures growing at higher rates. It was found that relief of the glucose limitation resulted in a shift in fermentation pattern: at the lower growth rates 50% or more of the extra glucose taken up was fermented to D-lactate.Incubation experiments with sonified cells revcaled that K. aerogenes possessed all the enzymes needed to convert dihydroxyacetone phosphate to methylglyoxal and subsequently to D-lactate, and that the rate at which this overall conversion occurred in vitro was in close agreement with the production rate of D-lactate in vivo. Moreover, it was found that the activities of the enzymes of the methylglyoxal bypass were dependent on the imposed growth rate. At higher growth rates, where cells possessed the potential to increase their growth rate immediately, the activity of methylglyoxal synthase was relatively low.it could be shown that, under low growth rate conditions, the uncoupling effect of the methylglyoxal bypass was highly effective and that, as a consequence thereof, a significant increase in the uptake rate of the energy source was accompanied by only a marginal increase in the rate at which ATP was synthesized.     

Expression of glf Z.m. increases D-mannitol formation in whole cell biotransformation with resting cells of Corynebacterium glutamicum

A recombinant oxidation/reduction cycle for the conversion of D-fructose to D-mannitol was established in resting cells of Corynebacterium glutamicum. Whole cells were used as biocatalysts, supplied with 250 mM sodium formate and 500 mM D-fructose at pH 6.5. The mannitol dehydrogenase gene (mdh) from Leuconostoc pseudomesenteroides was overexpressed in strain C. glutamicum ATCC 13032. To ensure sufficient cofactor [nicotinamide adenine dinucleotide (reduced form, NADH)] supply, the fdh gene encoding formate dehydrogenase from Mycobacterium vaccae N10 was coexpressed. The recombinant C. glutamicum cells produced D-mannitol at a constant production rate of 0.22 g (g cdw)⁻¹ h⁻¹. Expression of the glucose/fructose facilitator gene glf from Zymomonas mobilis in C. glutamicum led to a 5.5-fold increased productivity of 1.25 g (g cdw)⁻¹ h⁻¹, yielding 87 g l⁻¹ D-mannitol from 93.7 g l⁻¹ D-fructose. Determination of intracellular NAD(H) concentration during biotransformation showed a constant NAD(H) pool size and a NADH/NAD⁺ ratio of approximately 1. In repetitive fed-batch biotransformation, 285 g l⁻¹ D-mannitol over a time period of 96 h with an average productivity of 1.0 g (g cdw)⁻¹ h⁻¹ was formed. These results show that C. glutamicum is a favorable biocatalyst for long-term biotransformation with resting cells. Dedicated to Prof. Hermann Sahm on the occasion of his 65th birthday.     

Influence of the glucose input concentration on the kinetics of metabolite production by Klebsiella aerogenes NCTC 418: Growing in chemostat culture in potassium- or ammonia-limited environments

Thiobacillus neapolitanus grown in minerals medium in a thiosulfate-limited chemostat excreted 15% of all the carbon dioxide fixed as ¹⁴C-organic compounds at a dilution rate (D) of 0.03 h^-1. At D=0.36 h^-1 this excretion was 8.5%. Up to a D of 0.2h^-1 glycolate was the major excretion product. Glycolate excretion was maximal at a pO₂ of 100% air saturation (a.s.) and not detectable at a pO₂ of 5% (a.s.). Increasing the pCO₂ of the gassing mixture to 5% (v/v), at a pO₂ of 50% a.s. resulted in a lowering of the glycolate excretion from 3.5% of the total CO₂ fixed to 1.8%. These results indicate that glycolate excretion in T. neapolitanus is due to oxygenase activity of D-ribulose-1,5-bisphosphate carboxylase. HPMS (2-pyridylhydroxymethanesulfonate), an inhibitor of glycolate metabolism, did not stimulate the glycolate production in T. neapolitanus. Glycolate excretion was not observed in thiosulfate-limited chemostat cultures of the obligately chemolithotrophic Thiomicrospira pelophila or in thiosulfate- or formate-grown cultures of the facultatively chemolithotrophic Thiobacillus A2.Abbreviation HPMS 2-pyridylhydroxymethanesulfonate     

Conversion of glycerol to pyruvate by Escherichia coli using acetate- and acetate/glucose-limited fed-batch processes

We report the conversion of glycerol to pyruvate by E. coli ALS929 containing knockouts in the genes encoding for phosphoenolpyruvate synthase, lactate dehydrogenase, pyruvate formate lyase, the pyruvate dehydrogenase complex, and pyruvate oxidase. As a result of these knockouts, ALS929 has a growth requirement of acetate for the generation of acetyl CoA. In steady-state chemostat experiments using excess glycerol and limited by acetate, lower growth rates favored the formation of pyruvate from glycerol (0.60 g/g at 0.10 h⁻¹ versus 0.44 g/g at 0.25 h⁻¹), while higher growth rates resulted in the maximum specific glycerol consumption rate (0.85 g/g h at 0.25 h⁻¹ versus 0.59 g/g h at 0.10 h⁻¹). The presence of glucose significantly improved pyruvate productivity and yield from glycerol (0.72 g/g at 0.10 h⁻¹). In fed-batch studies using exponential acetate/glucose-limited feeding at a constant growth rate of 0.10 h⁻¹, the final pyruvate concentration achieved was about 40 g/L in 36 h. A derivative of ALS929 which additionally knocked out methylglyoxal synthase did not further increase pyruvate productivity or yield, indicating that pyruvate formation was not limited by accumulation of methylglyoxal.     

The effect of levofloxacin concentration on the development and maintenance of antibiotic-resistant clones of Escherichia coli in chemostat culture

A levofloxacin-sensitive strain of Escherichia coli (broth MIC: 0.0625 mg l⁻¹) was grown in carbon-limited chemostat culture for 316 h (D=0.294 h⁻¹). Hyperresistant strains isolated after 58 and 91 generations of culture retained a 16- to 47-fold increase in tolerance to levofloxacin during antibiotic-free serial batch and continuous culture (20 generations, glucose-limited, D=0.2 h⁻¹). Isolates differed from the original strain in their maximum growth rates in the presence and absence of subinhibitory levels of levofloxacin, protein-banding profiles, and resistance to a range of antibiotics. Competition between resistant isolates and the original sensitive strain was studied in glucose-limited chemostat cultures (D=0.2 h⁻¹). At levofloxacin concentrations less than 0.03 mg l⁻¹, the sensitive strain outcompeted resistant isolates and displaced them from the culture, whereas the reverse was true at higher concentrations. These results have clinical and environmental implications for those administering levofloxacin. Journal of Industrial Microbiology & Biotechnology (2002) 29, 155–162 doi:10.1038/sj.jim.7000295 Received 26 September 2001/ Accepted in revised form 13 June 2002     

Continuous fermentation to produce xanthan biopolymer: Effect of dilution rate

Single-stage continuous fermentations to produce xanthan gum have been run at dilution rates (D) from 0.023 to 0.196 hr^?1. Xanthan production rate (XPR) was a function of D. XPR increased from 0.34 g/hr/kg at D = 0.023 hr^?1 to the maximum 0.84 g/hr/kg at D = ca. 0.15 hr^?1. At D > 0.15 hr^?1 XPR decreased and at the highest D studied (0.196 hr^?1) was 0.69 g/hr/kg. Yield of xanthan from glucose consumed was 81–89%. Steady states ended between 6.5 and 8.7 turnovers when a variant strain occurred.     

Energetics of mixotrophic and autotrophic C1-metabolism by Thiobacillus acidophilus

Although the facultatively autotrophic acidophile Thiobacillus acidophilus is unable to grow on formate and formaldehyde in batch cultures, cells from glucose-limited chemostat cultures exhibited substrate-dependent oxygen uptake with these C₁-compounds. Oxidation of formate and formaldehyde was uncoupler-sensitive, suggesting that active transport was involved in the metabolism of these compounds. Formate- and formaldehyde-dependent oxygen uptake was strongly inhibited at substrate concentrations above 150 and 400 M, respectively. However, autotrophic formate-limited chemostat cultures were obtained by carefully increasing the formate to glucose ratio in the reservoir medium of mixotrophic chemostat cultures. The molar growth yield on formate (Y=2.5 g ·mol^-1 at a dilution rate of 0.05 h^-1) and RuBPCase activities in cell-free extracts suggested that T. acidophilus employs the Calvin cycle for carbon assimilation during growth on formate. T. acidophilus was unable to utilize the C₁-compounds methanol and methylamine. Formate-dependent oxygen uptake was expressed constitutively under a variety of growth conditions. Cell-free extracts contained both dye-linked and NAD-dependent formate dehydrogenase activities. NAD-dependent oxidation of formaldehyde required reduced glutathione. In addition, cell-free extracts contained a dye-linked formaldehyde dehydrogenase activity. Mixotrophic growth yields were higher than the sum of the heterotrophic and autotrophic yields. A quantitative analysis of the mixotrophic growth studies revealed that formaldehyde was a more effective energy source than formate.     

Succinic acid-producing biofilms of Actinobacillus succinogenes: reproducibility,stability and productivity

Continuous anaerobic fermentations were performed in a biofilm reactor packed with Poraver® beads. Dilution rates (D) varied between 0.054 and 0.72 h^?1, and d-glucose and CO₂ gas were used as carbon substrates. Steady-state conditions were shown to be repeatable and independent of the operational history. Production stability was achieved over periods exceeding 80 h at values of D below 0.32 h^?1. In these situations, steady-state variation (expressed as fluctuations in NaOH neutralisation flow rates) exhibited a standard deviation of less than 5 % while no indication of biofilm deactivation was detected. The total biomass amount was found to be independent of the dilution rate with an average dry concentration of 23.8?±?2.9 g L^?1 obtained for all runs. This suggests that the attachment area controls the extent of biofilm accumulation. Specific succinic acid (SA) productivities, based on the total biomass amount, exhibited a substantial decrease with decreasing D. An SA volumetric productivity of 10.8 g L^?1 h^?1 was obtained at D?=?0.7 h^?1—the highest value reported to date in Actinobacillus succinogenes fermentations. SA yields on glucose increased with decreasing D, with a yield of 0.90?±?0.01 g g^?1 obtained at a D of 0.054 h^?1. Production of formic acid approached zero with decreasing D, while the succinic to acetic acid ratio increased with decreasing D, resulting in an increasing SA yield on glucose.     

Cytosolic NADPH balancing in <Emphasis Type="Italic">Penicillium chrysogenum</Emphasis> cultivated on mixtures of glucose and ethanol

The in vivo flux through the oxidative branch of the pentose phosphate pathway (oxPPP) in Penicillium chrysogenum was determined during growth in glucose/ethanol carbon-limited chemostat cultures, at the same growth rate. Non-stationary ¹³C flux analysis was used to measure the oxPPP flux. A nearly constant oxPPP flux was found for all glucose/ethanol ratios studied. This indicates that the cytosolic NADPH supply is independent of the amount of assimilated ethanol. The cofactor assignment in the model of van Gulik et al. (Biotechnol Bioeng 68(6):602–618, 2000) was supported using the published genome annotation of P. chrysogenum. Metabolic flux analysis showed that NADPH requirements in the cytosol remain nearly the same in these experiments due to constant biomass growth. Based on the cytosolic NADPH balance, it is known that the cytosolic aldehyde dehydrogenase in P. chrysogenum is NAD⁺ dependent. Metabolic modeling shows that changing the NAD⁺-aldehyde dehydrogenase to NADP⁺-aldehyde dehydrogenase can increase the penicillin yield on substrate.