Substrate Consumption Rate Research Articles

Fermentation of glucose/xylose mixtures using Pichia stipitis

Different synthetic glucose/xylose mixtures (60 g/L total sugar) were fermented into ethanol using P. stipitis CBS 6054. Glucose was the preferred substrate in the glucose/xylose mixtures. The high glucose fractions had higher cell biomass production rate and therefore higher substrate consumption rate and ethanol production rate compared to high xylose fractions. However, the high xylose fractions had a slightly higher ethanol yields compared to the high glucose fractions because the substrate was channeled into ethanol production rather the cell biomass. The maximum ethanol concentration was 22.7 and 24.3 g/L in 60 g/L glucose and xylose media, respectively. Using the same initial cell concentration of 2 g/L, fermentation was complete after 96 h of fermentation in 100% glucose (60 g/L), and after 120 h of fermentation in 100% xylose (60 g/L).

Application of 13C-[2] - and 13C-[1,2] acetate in metabolic labelling studies of yeast and insect cells

The advantage of using 13C-labelled glucose in metabolic studies is that it is an important carbon and energy source for almost all biotechnologically and medically important organisms. On the other hand, the disadvantage is its relatively high cost in the labelling experiments. Looking for cheaper alternatives we found that 13C-[2] acetate or 13C-[1,2] acetate is a prospective compound for such experiments. Acetate is well incorporated by many organisms, including mammalian and insect cell cultures as preferred source of acetyl-CoA. Our experimental results using 13C NMR demonstrated that acetate was efficiently incorporated into glutamate and alanine secreted by the insect cell culture. Using D-stat culture of Saccharomyces uvarum on glucose/13C-acetate mineral media we demonstrated that the labelling patterns of proteinogenic amino acids can be well predicted on the basis of specific substrate consumption rates using the modified scheme of yeast metabolism and stoichiometric modelling. According to this scheme aspartate and alanine in S. uvarum under the experimental conditions used is synthesised in the mitochondria. Synthesis of alanine in the mitochondria was also demonstrated for Spodoptera frugiperda. For both organisms malic enzyme was also operative. For S. uvarum it was shown that the activity of malic enzyme is sufficient for supporting the mitochondrial biosynthetic reactions with NADPH.

Kinetics of High Cell Density Fed-batch Culture of Recombinant Escherichia coli Producing Human-like Collagen

The kinetics of batch and fed-batch cultures of recombinant Escherichia coli producing human-like collagen was investigated. In the batch culture, a kinetic model of a simple growth-association system was concluded without consideration of cell endogeneous metabolism. The cell lag time, the maximum specific growth rate and Y X/S were determined as 1.75h, 0.65h −1 and 0.51g·g −1, respectively. In the fed-batch culture, different specific growth rates were set at (0.15, 0.2, 0.25h −1) by the method of pseudo-exponential feeding, and the expressions for the specific rate of substrate consumption, the growth kinetics and the product formation kinetics of each phase were obtained. The result shows that the concentrations of cell and product can reach 77.5g·L −1 and 10.2g·L −1 respectively. The model predictions are in good agreement with the experimental data.

Lactic acid production from xylose by the fungus Rhizopus oryzae

Lignocellulosic biomass is considered nowadays to be an economically attractive carbohydrate feedstock for large-scale fermentation of bulk chemicals such as lactic acid. The filamentous fungus Rhizopus oryzae is able to grow in mineral medium with glucose as sole carbon source and to produce optically pure L(+)-lactic acid. Less is known about the conversion by R. oryzae of pentose sugars such as xylose, which is abundantly present in lignocellulosic hydrolysates. This paper describes the conversion of xylose in synthetic media into lactic acid by ten R. oryzae strains resulting in yields between 0.41 and 0.71 g g(-1). By-products were fungal biomass, xylitol, glycerol, ethanol and carbon dioxide. The growth of R. oryzae CBS 112.07 in media with initial xylose concentrations above 40 g l(-1) showed inhibition of substrate consumption and lactic acid production rates. In case of mixed substrates, diauxic growth was observed where consumption of glucose and xylose occurred subsequently. Sugar consumption rate and lactic acid production rate were significantly higher during glucose consumption phase compared to xylose consumption phase. Available xylose (10.3 g l(-1)) and glucose (19.2 g l(-1)) present in a mild-temperature alkaline treated wheat straw hydrolysate was converted subsequently by R. oryzae with rates of 2.2 g glucose l(-1) h(-1) and 0.5 g xylose l(-1) h(-1). This resulted mainly into the product lactic acid (6.8 g l(-1)) and ethanol (5.7 g l(-1)).

Rhizopus oryzae fungus cells producing L(+)-lactic acid: kinetic and metabolic parameters of free and PVA-cryogel-entrapped mycelium

Spores of the filamentous fungus Rhizopus oryzae were entrapped in macroporous poly(vinyl alcohol) cryogel (PVA-cryogel). To prepare immobilised biocatalyst capable of producing L(+)-lactic acid (LA), the fungus cells were cultivated inside the carrier beads. The growth parameters and metabolic activity of the suspended (free) and immobilised cells producing LA in a batch process were comparatively investigated. The immobilised cells possessed increased resistance to high concentrations of accumulated product and gave much higher yields of LA in the iterative working cycle than the free cells did. Detailed kinetic analysis of the changes in the intracellular adenosine triphosphate concentration, specific rate of growth, substrate consumption and LA production showed that the fungus cells entrapped in PVA-cryogel are more attractive for biotechnological applications compared to the free cells.

Aspergillus niger ATCC 9142 세포에 의해 농축된 우유공장폐수로부터 구연산생산에 대한 동력학 연구

Aspegillus niger 세포에 의한 구연산 생산에 대한 발효 매체로서 우유공장폐수가 연구되었다. 발효매체에 <TEX>$Mn^{2+},\;Fe^{2+}\;and\;Cu^{2+}$</TEX>의 첨가가 구연산 생산을 일정하게 증가시켰다. 그러나 다른 금속이온인 <TEX>$Mg^{2+}$</TEX>의 첨가는 구연산 생산을 감소시켰다. 구연산의 농도는 우유공장폐수에서 환원당 농도의 농도가 50 g/l와 100 g/1로 Aspegillus niger ATCC 9142에 의한 batch bioreactor에서 각각 7.2 g/1과 16.5 g/l로 높게 표시되었다. 본 실험에서 적용한 수리적 모델식들이 세포성장의 예상, 구연산 생산, 기질소모 속도에 대해 잘 적용되는지를 알아보기 위해서 발효반응 모델식에 적용시켜 본 결과 실험치와 잘 부합되었다. The waste water from a milk factory was investigated for possibility of use to the production of citric acid by cells of Aspegillus niger ATCC 9142. The addition of <TEX>$Mn^{2+},\;Fe^{2+}\;and\;Cu^{2+}$</TEX> ions to waste increased citric acid production steadily, but addition of metal ion <TEX>$Mg^{2+}$</TEX> decreased the citric acid production. The amount of produced citric acid by Aspegillus niger ATCC 9142 with addition 50 g/1 and 100 g/1 of reducing sugar in milk factory waste water were 7.2 g/1 and 16.5 g/1 respectively. Mathematical model was simulated for their predictability of cell growth, citric acid production and substrate consumption rate and coincided with experimental data.

Species-independent maintenance energy and natural population sizes

A general strategy to estimate the size of an active microbial community based on catabolic rates was developed. Tijhuis et al. (Biotechnol. Bioeng. 42, 1993, 509–519) calculated from chemostat experiments maintenance energy consumption rates by multiplication of the maintenance substrate consumption rate with the standard free energy of the catabolic reaction. They showed that the maintenance energy can generally be described as species-independent by an Arrhenius equation. In the present communication, it is shown that the theory is applicable for different microbial culture systems. For a given environmental situation, the maximal microbial population size is obtained when the free energy conserved is consumed solely for maintenance of the cell. The free energy conservation rate per volume together with the species-independent description of maintenance energy enables calculations of the maximal population size corresponding to a catabolic rate and of maintenance substrate consumption rates for microbial cultures.

Interactions Between Carbon Nanotubes and Bacteria

ABSTRACTWhile carbon nanotubes (CNTs) possess diverse application potential ranging from polymer nanocomposites to nanostructured therapeutic devices, their environmental implications are still not well understood. In this study, we conducted experiments to monitor changes in bacterial physiology as a consequence of interactions with the CNTs. Our experiments included a common anaerobe,Escherichia coli, in an SWNT system as a model for understanding the environmental implications of CNTs. Observations over several weeks indicated that the bacteria in the presence of SWNTs showed significant morphological changes, that included elongation. It is interesting to note that similar morphological changes have been reported in response to extreme temperature and pressure, chemical agents, and quantum dots. In this presentation, the authors will present further physiological evidence, such as bacterial growth and substrate consumption rates, towards defining a possible bacteria-CNT interaction model consistent with these experimental observations, and will discuss the overall environmental implications.

Control of catalytic debenzylation and dehalogenation reactions during liquid-phase reduction by H 2

Heterogeneous catalysis, which is being used more frequently in the fine chemicals industry, does not always provide the desired selectivity because various functional groups become labile to reduction by hydrogen on a noble metal under similar reaction conditions. To study the selectivity of debenzylation versus dechlorination in an aromatic compound, the kinetics of the reduction of 4-chloro-N,N-dibenzylaniline on supported Pd catalysts in a buffered solvent under hydrogen reduction conditions were investigated. The turnover frequency on a Pd/C catalyst was 2–40 times higher than that on Pd dispersed on SiO 2, TiO 2, and Al 2O 3. The effects of hydrogen pressure, organic substrate concentration, temperature, solvent, and catalyst support on the selectivity were not great at a pH of 5.4, whereas the effect of acid or base modifiers was very significant. Only dechlorination reactions occurred under basic conditions, and the use of an acid confirmed a preferential promotion of debenzylation reactions; consequently, as the pH decreased from 12 to 0.1, the selectivity for dechlorination decreased from essentially 100% to almost 0. Using a triethylamine/acetic acid buffer system to control pH, it was determined that the inflection point in selectivity corresponds to a pH value equal to the p K a of the benzyl-protected amine (4.5 in this case). Consequently, one important finding is that the dechlorination reaction can occur at high rates even under acidic conditions, as long as the pH of the reaction mixture is greater than the p K a of the protected amine. Furthermore, generation of an acidic product like HCl during a dechlorination reaction can decrease the system pH and markedly alter selectivity unless a buffer is present. For reactions carried out at a pH of 5.4, the initial rate of organic substrate consumption was modeled by a classical Langmuir–Hinshelwood sequence, with the assumption of two types of active sites, one site to adsorb H atoms and the other to adsorb the organic substrate, yielding a slightly better fit to the data. The Weisz–Prater criterion was applied to verify the absence of mass transfer effects.

Diffusion-Kinetic Rate of Substrate Consumption in a Biofilm

The diffusion-kinetic rate of substrate consumption by microorganisms in a biofilm is analyzed. Specific features of modes following from the Michaelis-Menten kinetic scheme are demonstrated with allowance for the dependence of the biomass concentration on the substrate concentration field.

Kinetics of Enhanced Substrate Consumption and Endo-β-xylanase Production by a Mutant Derivative of Humicola lanuginosa in Solid-state Fermentation

Industrial byproducts namely canola meal, rice bran, sunflower meal, and wheat straw were used as substrates for endo-xylanase production by Humicola lanuginosemutant TH1 through solid substrate fermentation. The enzyme was secreted extracellularly by both wild and mutant cultures. Rice bran supported the maximum production of endo-xylanase followed by wheat straw, canola meal and sunflower meal. The highest activity was achieved after 72 h of culture and the highest yields from the above substrates were 842, 840, 610 and 608 IU per g substrate consumed respectively. The highest productivity (281 IU flask−1 h−1 corresponding to 5620 l−1 h-1) of endo-xylanase by the mutant of H. lanuginosa was 1.6-fold more than that produced by the parental organism in solid-state fermentation of rice bran at 45 °C. Maximum specific activity (180 IU mg−1 protein) and substrate consumption rates were significantly more than those reported by previous researchers on Humicola sp. The mutant possessed markedly low accompanying cellulase activity. Thermodynamic studies revealed that the mutant required significantly lower activation energy for enzyme production and higher for thermal inactivation which signified that the endogenous metabolic machinery of mutant cells exerted more protection against thermal inactivation during product formation than that needed by its parental cultures.

Denitrification potential of industrial wastewaters

Nitrate utilisation batch experiments have been carried out with different agro-food industrial wastewaters. The results obtained were modelled using a modification of the ASM2d to determine the main kinetic and stoichiometric parameters: denitrification rate with fermentation products ( q N−SA) and with fermentable substrates ( q N−SF); fermentation products and fermentable substrates consumption rates, ( q SA) and ( q SF), respectively; yield coefficient with fermentation products ( Y H−SA) and with fermentable substrates ( Y H−SF) and saturation coefficient for growth on fermentation products ( K H−SA). It was found that fermentation products ( S A) and fermentable substrates ( S F) consumption rates can be assumed to be both constant and independent of the agro-food industrial wastewater used, with values of 14.6 and 5.4 (mg S COD g COD- X OHO –1 h –1), respectively. The mean denitrification rates obtained with each agro-food industrial wastewater ranged from 1.5 to 2.5 (mg S N g COD- X OHO –1 h –1), values slightly lower to the obtained with domestic wastewater (2.7 mg S N g COD- X OHO –1 h –1). Given the substrate consumption rate and the denitrification rate, the yields with fermentation products and fermentable substrates for each wastewater were determined. The values obtained for the yields ranged from 0.50 to 0.69 on using S A and from 0.50 to 0.70 on using S F. Having determined these parameters, the denitrification potential (DNP) was evaluated for each agro-food wastewater. Wastewater from slaughterhouses gave the lowest DNP (22.6 mg S N g S COD –1), whereas wastewater from potato and tomato processing plants gave the highest values (126.5 and 128.2 mg S N g S COD –1, respectively), which are very similar to that obtained with domestic wastewater (130.3 and 128.2 mg S N g S COD –1). Taking into account the low N/COD ratio presented in the agro-food wastewaters, the last wastewaters are the most indicated to enhance the nitrogen removal in the WWTP. Finally, a relationship between the denitrification rates of several agro-food industrial wastewaters and their respective S A/( S A+ S F) ratio was obtained. This relationship can be used to estimate the mean denitrification rate of an agro-food industrial wastewater.

Aromatic and aliphatic hydrocarbon consumption and transformation by the styrene degrading strain Pseudomonas putida CA-3

Pseudomonas putida CA-3 is capable of consuming a number of aromatic and aliphatic hydrocarbons. With the exception of styrene none of the alkenes tested are capable of supporting the growth of P. putida CA-3 as sole sources of carbon and energy. The highest rate of alkene consumption was observed with styrene as the substrate. A 6.5- and 15.5-fold lower rate of substrate consumption was observed with indene and indole with the concomitant formation of 2-indanone and indigo, respectively. The presence of a sulphur (benzothiopene) or oxygen (benzofuran) in the cyclopentene ring resulted in further decreases in the rate of substrate consumption by whole cells of P. putida CA-3. P. putida CA-3 is incapable of consuming benzene and consumes toluene at a low rate. No detectable products were observed in supernatants of cultures incubated with benzothiopene, benzofuran or toluene. The aliphatic alkenes 1-octene and 1,7-octadiene were both consumed by whole cells of P. putida CA-3 at a rate equivalent to indene consumption. The consumption of ( R) styrene oxide was 1.7- and 1.25-fold higher than that of the S isomer and the racemic mix, respectively. The rate of racemic indene oxide, 1,2-epoxyoctane and 1,2-epoxy-7-octene consumption was lower than their equivalent alkene and 55-, 11.8-, and 27.5-fold lower than the rate of racemic styrene oxide consumption. A transposon mutant incapable of growth with styrene or styrene oxide failed to transform indole to indigo. The ratio of styrene utilisation relative to other substrates changes in the mutant strain compared to the wild-type strain, e.g., Indene biotransformation by mutant AF5 is 1.9-fold higher than styrene consumption compared to the wild-type strain CA-3 where the rate of styrene consumption is 6.7-fold higher than indene consumption. This trend is also observed for other alkenes and epoxides.

Kinetic Characterization of Methanobacterium bryantii M.o.H.

H2 is a key electron donor for many anaerobic microorganisms; thus, keen competition for H2 occurs among H2-utilizing microbial groups. Monod kinetic parameters provide essential information for kinetic analysis of competition for H2. In this study, we estimated Monod kinetic parameter values for a methanogen that consumes only H2 as its electron donor, Methanobacterium bryantii M.o.H. Utilization of a single electron donor is an advantage in this study, because complications from alternate metabolic pathways are avoided. Using a set of batch experiments designed to provide the best estimates of each parameter, we obtained these values: maximum specific growth rate (mumax) = 0.77/ day, maximum substrate consumption rate (qmax) = 2.36 mol-H2/gcells/day, true yield (Y) = 0.325 gcell/mol H2, fraction of donor electrons to synthesis (fs degrees) = 0.03 e-cell/e- donor, half-maximum-rate substrate concentration (Ks) = 18 000 nM = 18 microM H2, and endogenous decay rate (b) = 0.088/ day. This self-consistent set of parameters indicates that, when H2 is not limiting, M. bryantii M.o.H. is a slow grower (low mumax) compared to other H2-oxidizing methanogens and sulfate reducers, and this is mainly due to its low true Y, not a low qmax. The relatively high Ks and b values suggest that M. bryantii also may not be a strong competitor when H2 is limiting.

Reliability of methods for the determination of specific substrate consumption rates in batch culture

Four methods for the calculation of specific substrate consumption rates in batch cultivation were compared. Calculations based on a simple mathematical model resulted in the most reliable rate predictions. The application and accuracy of a novel, Logarithmic Method for rate estimations directly from experimental data were also demonstrated.

Binding of Two Flaviolin Substrate Molecules, Oxidative Coupling, and Crystal Structure of Streptomyces coelicolor A3(2) Cytochrome P450 158A2

Cytochrome P450 158A2 (CYP158A2) is encoded within a three-gene operon (sco1206-sco1208) in the prototypic soil bacterium Streptomyces coelicolor A3(2). This operon is widely conserved among streptomycetes. CYP158A2 has been suggested to produce polymers of flaviolin, a pigment that may protect microbes from UV radiation, in combination with the adjacent rppA gene, which encodes the type III polyketide synthase, 1,3,6,8-tetrahydroxynaphthalene synthase. Following cloning, expression, and purification of this cytochrome P450, we have shown that it can produce dimer and trimer products from the substrate flaviolin and that the structures of two of the dimeric products were established using mass spectrometry and multiple NMR methods. A comparison of the x-ray structures of ligand-free (1.75 angstroms) and flaviolin-bound (1.62 angstroms) forms of CYP158A2 demonstrates a major conformational change upon ligand binding that closes the entry into the active site, partly due to repositioning of the F and G helices. Particularly interesting is the presence of two molecules of flaviolin in the closed active site. The flaviolin molecules form a quasi-planar three-molecule stack including the heme of CYP158A2, suggesting that oxidative C-C coupling of these phenolic molecules leads to the production of flaviolin dimers.

Native specific activity of glutathione peroxidase (GPx‐1), phospholipid hydroperoxide glutathione peroxidase (PHGPx) and glutathione reductase (GR) does not differ between normo‐ and hypomotile human sperm samples

Native specific activity of glutathione peroxidase (GPx‐1), phospholipid hydroperoxide glutathione peroxidase (PHGPx) and glutathione reductase (GR) does not differ between normo‐ and hypomotile human sperm samples

Activity and Diversity of Methanogens in a Petroleum Hydrocarbon-Contaminated Aquifer

Methanogenic activity was investigated in a petroleum hydrocarbon-contaminated aquifer by using a series of four push-pull tests with acetate, formate, H(2) plus CO(2), or methanol to target different groups of methanogenic Archaea. Furthermore, the community composition of methanogens in water and aquifer material was explored by molecular analyses, i.e., fluorescence in situ hybridization (FISH), denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes amplified with the Archaea-specific primer set ARCH915 and UNI-b-rev, and sequencing of DNA from dominant DGGE bands. Molecular analyses were subsequently compared with push-pull test data. Methane was produced in all tests except for a separate test where 2-bromoethanesulfonate, a specific inhibitor of methanogens, was added. Substrate consumption rates were 0.11 mM day(-1) for methanol, 0.38 mM day(-1) for acetate, 0.90 mM day(-1) for H(2), and 1.85 mM day(-1) for formate. Substrate consumption and CH(4) production during all tests suggested that at least three different physiologic types of methanogens were present: H(2) plus CO(2) or formate, acetate, and methanol utilizers. The presence of 15 to 20 bands in DGGE profiles indicated a diverse archaeal population. High H(2) and formate consumption rates agreed with a high diversity of methanogenic Archaea consuming these substrates (16S rRNA gene sequences related to several members of the Methanomicrobiaceae) and the detection of Methanomicrobiaceae by using FISH (1.4% of total DAPI [4',6-diamidino-2-phenylindole]-stained microorganisms in one water sample; probe MG1200). Considerable acetate consumption agreed with the presence of sequences related to the obligate acetate degrader Methanosaeata concilii and the detection of this species by FISH (5 to 22% of total microorganisms; probe Rotcl1). The results suggest that both aceticlastic and CO(2)-type substrate-consuming methanogens are likely involved in the terminal step of hydrocarbon degradation, while methanogenesis from methanol plays a minor role. DGGE profiles further indicate similar archaeal community compositions in water and aquifer material. The combination of hydrogeological and molecular methods employed in this study provide improved information on the community and the potential activity of methanogens in a petroleum hydrocarbon-contaminated aquifer.

Mole Fraction Control of Poly([R]‐3‐hydroxybutyrate‐co‐3‐hydroxyvalerate) (PHB/HV) Synthesized by Paracoccus denitrificans

AbstractDifferent substrate mixtures of acetic acid and valeric acid were used to synthesize copolymers of poly([R]‐3‐hydroxybutyrate‐co‐3‐hydroxyvalerate) (PHB/HV) with Paracoccus denitrificans under N‐limited conditions. A correlation between the substrate ratio and HV content was found in batch experiments, which seemed to be suitable to produce a number of defined copolymers. In fed‐batch fermentation, such correlation could only be found with carbon substrate mixtures of very restricted composition. Due to the individual substrate consumption rates with this technique, a polymer with 16.5 mol.‐% HV content [w/w] could be reproducibly synthesized. However, under N‐limited chemostatic cultivation conditions it was possible to produce a spectrum of definitely composed copolymers (3.0 %–46.3 mol.‐% HV) from different mixtures of acetic acid and valeric acid.

Prolonged maltose-limited cultivation of Saccharomyces cerevisiae selects for cells with improved maltose affinity and hypersensitivity.

Prolonged cultivation (>25 generations) of Saccharomyces cerevisiae in aerobic, maltose-limited chemostat cultures led to profound physiological changes. Maltose hypersensitivity was observed when cells from prolonged cultivations were suddenly exposed to excess maltose. This substrate hypersensitivity was evident from massive cell lysis and loss of viability. During prolonged cultivation at a fixed specific growth rate, the affinity for the growth-limiting nutrient (i.e., maltose) increased, as evident from a decreasing residual maltose concentration. Furthermore, the capacity of maltose-dependent proton uptake increased up to 2.5-fold during prolonged cultivation. Genome-wide transcriptome analysis showed that the increased maltose transport capacity was not primarily due to increased transcript levels of maltose-permease genes upon prolonged cultivation. We propose that selection for improved substrate affinity (ratio of maximum substrate consumption rate and substrate saturation constant) in maltose-limited cultures leads to selection for cells with an increased capacity for maltose uptake. At the same time, the accumulative nature of maltose-proton symport in S. cerevisiae leads to unrestricted uptake when maltose-adapted cells are exposed to a substrate excess. These changes were retained after isolation of individual cell lines from the chemostat cultures and nonselective cultivation, indicating that mutations were involved. The observed trade-off between substrate affinity and substrate tolerance may be relevant for metabolic engineering and strain selection for utilization of substrates that are taken up by proton symport.