High Dilution Rates Research Articles

Nitrite-oxidizing bacteria guild ecology associated with nitrification failure in a continuous-flow reactor

Nitrification is an important process for nitrogen removal in many wastewater treatment plants, which requires the mutualistic oxidation of ammonia to nitrate by ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB). However, this process can be quite unpredictable because both guilds are conditionally sensitive to small changes in operating conditions. Here, dynamics are examined within the NOB guild in two parallel chemostats operated at low and high dilution rates (0.10 and 0.83 day(-1), respectively) during periods of varying nitrification performance. NOB and AOB guild abundances and nitrogen-oxidation efficiency were relatively constant over time in the 0.10 day(-1) reactor; however, the 0.83 day(-1) reactor had two major disturbance episodes that caused destabilization of the NOB guild, which ultimately led to nitrification failure. The first episode caused the extinction of Nitrospira spp. from the system, resulting in chronic incomplete ammonia oxidation and nitrite accumulation. The second episode caused complete loss of nitrification activity, likely resulting from metal toxicity and the previous extinction of Nitrospira spp. from the system. These results exemplify the types of changes that can occur within the NOB guild that result in process impairment or failure, and provide one possible explanation for why nitrification is often unstable at higher dilution rates.

Kinetic Measurement of Biological Oxidation of Ferrous Iron at Low Ferric to Ferrous Ratios in a Controlled Potential Batch Reactor

Traditionally, the kinetics of microbial ferrous iron oxidation have been studied in continuous culture or in batch. Both methods have drawbacks: in continuous culture experiments have to be repeated at a number of dilution rates to cover the entire spectrum of ferrous to ferric ratios, which is time-consuming. Furthermore, experiments at very low ferric to ferrous ratios generally fail due to microbial wash-out at the high dilution rates needed to achieve these. In batch experiments, on the other hand, the prevalent ferric to ferrous ratio rapidly changes due to substrate depletion while the microbial population continually grows, making determination of specific momentary rates difficult. The present paper describes initial work with a novel device, the Redostat™, which allows careful electrochemical control of ferric to ferrous ratio in a batch reactor. A culture of Leptospirillum ferriphilum was grown at 35°C and 5 g/L total iron by maintaining the ferric to ferrous ratio at 0.17, 0.51 and 1.65 (corresponding to redox potentials of 419, 452 and 482 mV vs. Ag/AgCl), respectively. The correlation of data obtained from off-gas and current measurements was excellent, and fitted Monod kinetics with ferric inhibition. A hitherto unobserved effect indicates the onset of ferric iron inhibition at the low redox potentials employed here.

The performance of serial bioreactors in rapid continuous production of ethanol from dilute-acid hydrolyzates using immobilized cells

The performance of single, and series of, continuous stirred-tank (CSTBR) and fluidized-bed bioreactor (FBBR) in anaerobic continuous cultivation of glucose in defined media and dilute-acid hydrolyzates at dilution rates 0.22, 0.43, 0.65 and 0.86 h −1 using immobilized Saccharomyces cerevisiae CBS 8066, was investigated. While the single CSTBR and FBBR could not take up more than 77% and 92% of glucose in a defined medium at dilution rate 0.86 h −1, addition of the second bioreactor decreased the residual glucose to less than 1.1% of the incoming sugar. A similar trend was obtained in cultivation of dilute-acid hydrolyzates. A CSTBR could take up 75% and 54% of the initial fermentable sugars at dilution rates 0.43 and 0.86 h −1, while the addition of the FBBR improved the assimilation of the sugars to 100% and 86%, respectively. The ethanol yields from the hydrolyzate were between 0.41 and 0.48 g/g in all the experiments. The specific and volumetric ethanol productivities were 1.13 g/g h and 5.98 g/L h for the single bioreactor and 0.98 g/g h and 5.49 g/L h for the serial bioreactor at the highest dilution rate, respectively. Glycerol was the only important by-product in terms of concentration, and yielded 0.05–0.07 g/g from the hydrolyzate. From the initial 3.98 g/L acetic acid present in the hydrolyzate, 0.1–0.8 g/L was assimilated by the cells. The yeast cells were accumulated close to the surface of the beads. While the cells had a dry-weight concentration of 129 g/L close to the surface of the beads, the concentration in the core was only 13 g/L.

Glucose uptake and growth of glucose-limited chemostat cultures of Aspergillus niger and a disruptant lacking MstA, a high-affinity glucose transporter

This is a study of high-affinity glucose uptake in Aspergillus niger and the effect of disruption of a high-affinity monosaccharide-transporter gene, mstA. The substrate saturation constant (K(s)) of a reference strain was about 15 microM in glucose-limited chemostat culture. Disruption of mstA resulted in a two- to fivefold reduction in affinity for glucose and led to expression of a low-affinity glucose transport gene, mstC, at high dilution rate. The effect of mstA disruption was more subtle at low and intermediate dilution rates, pointing to some degree of functional redundancy in the high-affinity uptake system of A. niger. The mstA disruptant and a reference strain were cultivated in glucose-limited chemostat cultures at low, intermediate and high dilution rate (D=0.07 h(-1), 0.14 h(-1) and 0.20 h(-1)). Mycelium harvested from steady-state cultures was subjected to glucose uptake assays, and analysed for expression of mstA and two other transporter genes, mstC and mstF. The capacity for glucose uptake (v(max)) of both strains was significantly reduced at low dilution rate. The glucose uptake assays revealed complex uptake kinetics. This impeded accurate determination of maximum specific uptake rates (v(max)) and apparent affinity constants ( ) at intermediate and high dilution rate. Two high-affinity glucose transporter genes, mstA and mstF, were expressed at all three dilution rates in chemostat cultures, in contrast to batch culture, where only mstC was expressed. Expression patterns of the three transporter genes suggested differential regulation and functionality of their products.

Effect of dilution rate on the microbial structure of a mesophilic butyrate-degrading methanogenic community during continuous cultivation

We constructed two mesophilic anaerobic chemostats that were continuously fed with synthetic wastewater containing butyrate as the sole source of carbon and energy. Steady-state conditions were achieved at dilution rates between 0.025 and 0.7 day(-1). Butyrate, fed into the chemostat, was almost completely mineralized to CH(4) and CO(2) at dilution rates below 0.5 day(-1). The butyrate-degrading methanogenic communities in the chemostats at dilution rates between 0.025 and 0.7 day(-1) were monitored based on the 16S rRNA gene, using molecular biological techniques including clone library analysis, denaturing gradient gel electrophoresis, and quantitative real-time polymerase chain reaction. The aceticlastic methanogen Methanosaeta and the hydrogenotrophic methanogen Methanoculleus dominated in methanogens at low dilution rates, whereas the aceticlastic methanogen Methanosaeta, Methanosarcina, the hydrogenotrophic methanogen Methanoculleus, and Methanospirillum dominated at high dilution rates. Bacteria affiliated with the family Syntrophaceae in the phylum Proteobacteria predominated at the low dilution rate of 0.025 day(-1), whereas bacteria affiliated with the phylum Firmicutes and Candidate division OP3 predominated at high dilution rates. A significant quantity of bacteria closely related to the genus Syntrophomonas was detected at high dilution rates. Dilution rate showed an apparent effect on archaeal and bacterial communities in the butyrate-fed chemostats.

Metabolic flux profiling of Pichia pastoris grown on glycerol/methanol mixtures in chemostat cultures at low and high dilution rates

The metabolic pathways associated with the tricarboxylic acid cycle intermediates of Pichia pastoris were studied using biosynthetically directed fractional 13C labelling. Cells were grown aerobically in a chemostat culture fed at two dilution rates (1.39x10(-5) s-1 and 4.44x10(-5) s-1) with varying mixtures of glycerol and methanol as sole carbon sources. The results show that, with co-assimilation of methanol, the common amino acids are synthesized as in P. pastoris cells grown on glycerol only. During growth at the lower dilution rate, when both substrates are entirely consumed, the incorporation of methanol into the biomass increases as the methanol fraction in the feed is increased. Moreover, the co-assimilation of methanol impacts on how key intermediates of the pentose phosphate pathway (PPP) are synthesized. In contrast, such an impact on the PPP is not observed at the higher dilution rate, where methanol is only partially consumed. This finding possibly indicates that the distribution of methanol carbon into assimilatory and dissimilatory (direct oxidation to CO2) pathways are different at the two dilution rates. Remarkably, distinct flux ratios were registered at each of the two growth rates, while the dependency of the flux ratios on the varying fraction of methanol in the medium was much less pronounced. This study brings new insights into the complex regulation of P. pastoris methanol metabolism in the presence of a second carbon source, revealing important implications for biotechnological applications.

Production of Solvent (acetone-butanol-ethanol) in Continuous Fermentation by Clostridium saccharobutylicum DSM 13864 Using Gelatinised Sago Starch as a Carbon Source

Solvent production by Clostridium saccharobutylicum DSM 13864 was carried out in a single stage continuous culture using 2 L stirred tank fermenter with gelatinised sago starch as a carbon source. From the study it was found that the condition could be adjusted to suit for acids production (high dilution rate and high pH) or solvent production (low dilution rate and low pH) by manipulating the dilution rate and culture pH of single stage continuous fermentation. The highest solvent concentration in outflow (9.10 g/L) was obtained at pH 4.5 and dilution rate of 0.05 h 1 , which corresponds to overall productivity of 0.46 g/L.h. However, the highest total solvent productivity (0.85 g/L.h) was obtained at dilution rate of 0.11 h -1 and pH 4.5, which gave a total solvent yield of 0.29 g solvent/g sago starch. Although the total solvent productivity was greatly increased in continuous culture, the final solvent concentration attained in outflow was decreased by about 53% as compared to batch culture.

Continuous hydrogen production by anaerobic mixed microflora using a hollow-fiber microfiltration membrane bioreactor

A membrane bioreactor (MBR) fabricated by connecting a hollow-fiber microfiltration membrane module with a continuous flow stirred tank reactor (CSTR) was used to enhance H 2 production through high-dilution rate operations. Three different carbon substrates (glucose, sucrose, and fructose) were examined for their effectiveness in H 2 production with a mixed microflora. The results show that in CSTR operation, cell washout occurred at a hydraulic retention time (HRT) of 2–4 h. Using MBR could avoid cell washout, leading to a substantial increase in both H 2 production rate (HPR) and biomass concentration. The MBR system was very effective in retaining biomass within the reactor as the system can be stably operated at an extremely low HRT of 1 h with an optimal steady-state HPR of 1.48, 2.07, and 2.75 l/h/l, respectively, for using glucose, sucrose, and fructose as the sole carbon source. Meanwhile, despite operation at a high dilution rate (i.e., HRT = 1 h ), the H 2 yield (HY) could be maintained at a high level of 1.27, 1.39, and 1.36 mol H 2 / mol hexose, for glucose, sucrose, and fructose, respectively. Irrespective of the bioreactor mode (CSTR or MBR), the HPR tended to decrease in the order of fructose > sucrose > glucose . Thus, fructose seems to be the most efficient carbon substrate for H 2 production with the H 2 -producing mixed culture used in this work. Butyrate (HBu) and acetate (HAc) (especially, HBu) were the major soluble metabolites in all cases, contributing to 70–85% of total soluble microbial product (SMP). The HPR and HY could be estimated based on stoichiometric correlation between formation of soluble metabolites (i.e., HBu, HAc, and propionate) and H 2 production. The estimated values are in good agreement with the experimental results.

Investigation of the response of separate electrodes in a polymer electrolyte membrane fuel cell without reference electrode

The paper presents electrochemical measurements carried out in a PEMFC with a view to determining the separate kinetics of the electrode reactions. For this purpose, the separate response of one electrode (anode or cathode) was magnified by dilution of the reacting gas, respectively hydrogen and oxygen, and comparison of the experimental data in the form of steady voltage-current variations and impedance spectra. Experiments were carried out at 60 °C and ambient pressure. Water management was thoroughly controlled so that the gases leaving the cell had the same relative humidity in all experiments of one series. Hydrogen oxidation, although rapid, corresponds to overpotentials up to 50 mV at high dilution rates and current densities. Assuming a Tafel–Volmer mechanism, the exchange current density of the anode reaction at the Pt surface is of the order of 1 mA cm−2. The two techniques employed led to Tafel slopes of oxygen reduction ranging from 120 to 150 mV/decade, with an exchange current density near 1 μA cm−2, in good agreement with published data.

OC4 Influence of Seminal Plasma Added to Highly Diluted Semen on Sperm Motility of Young Boar Feed with Selenium and Vitamin E

High dilution rates have been documented as detrimental for boar spermatozoa, shortening their lifespan (Centurion et al. 2003, Biol Reprod 69: 640–646). Addition of seminal plasma (SP) to semen extenders, or selenium (Se) and vitamin E (VE) in diet of boars could increase motility of highly diluted spermatozoa (HDS). The aim of this work was to evaluate the effect of seminal plasma on sperm motility of HDS from boars feed with Se and VE. Sixteen 12 month‐old boars were designed to one of four dietary treatments: (i) control, Se 0 ppm–VE 0 IU/kg; (ii) Se 0–VE 250; (iii) Se 0.5–VE 250 and (iv) Se 0.5–VE 0. Boars were treated for 8 weeks before semen collection. Sperm rich fractions from each boar were diluted to 5 × 106 sperm/ml in PBS medium and incubated at 37°C with or without 10% SP. The measurements were done at 0, 2 or 5 h. Data were analyzed as a mixed model for a factorial design [2 (Se) × 2 (VE) × 2 (SP) × 3 (h)]. Percentage of sperm motility (PSM) increases significantly (p < 0.001) with addition of Se (81.3 ± 1.52), VE (81.0 ± 1.62) and SP (81.5 ± 1.57) vs control (73.4 ± 1.61). There was significant interaction Se × VE (p < 0.001) and Se × VE × SP (p < 0.05) in PSM. However, PSM was affected significantly by time (0 h 83.4 ± 1.92; 2 h 80.7 ± 1.92 and 5 h 67.9 ± 1.92; p < 0.001). There was significant interaction SP × Time (p < 0.05) in PSM. These results indicate that Se, VE and SP improve seminal viability. Addition of 10% of SP maintains PSM at least during 5 hours.

Continuous Ethanol Fermentation Coupled with Recycling of Yeast Flocs

A continuous ethanol fermentation system composed of three-stage tanks in series coupled with two sedimentation tanks was established. A self-flocculating yeast strain developed by protoplast fusion from Saccharomyces cerevisiae and Schizosaccharomyces pombe was applied. Two-stage enzymatic hydrolysate of corn powder containing 220 g/L residual reducing sugar, supplemented with 1.5 g/L (NH 4) 2HPO 4 and 2.5 g/L KH 2PO 4, was used as the ethanol fermentation substrate and fed into the first fermentor at the dilution rate of 0.057 h −1. The yeast flocs separated by sedimentation were recycled into the first fermentor as two models: activation-recycle and direct-recycle. The quasi-steady states were obtained for both operation models after the fermentation systems experienced short periods of transitions. Activation process helped enhance the performance of ethanol fermentation at high dilution rates. The broth containing more than 101 g/L ethanol, 3.2 g/L residual reducing sugar, and 7.7 g/L residual total sugar was produced. The ethanol productivity was calculated to be 5.77 g L −1 h −1, which increased by more than 70 % compared with that achieved in the same tank in series system without recycling of yeast cells.

Comparison between the heterotrophic cultivation of Paracoccus denitrificans in continuous stirred tank reactor and horizontal rotating tubular bioreactor

Bacterium Paracoccus denitrificans is capable to obtain metabolic energy by oxidation of ammonia (chemolitotrophic metabolism) and organic compounds (chemoorganotrophic or heterotrophic metabolism). It can also perform nitrate and nitrite respiration. When cultivated in certain conditions P. denitrificans can conduct the complete transformation of ammonia into gaseous nitrogen and consequently the whole nitrogen removal process can be carried out in one step. These characteristics make it suitable for successful treatment of wastewater with high nitrogen content. In this work the heterotrophic cultivation of P. denitrificans was studied in continuous stirred tank reactor (CSTR) and in horizontal rotating tubular bioreactor (HRTB). Bioprocess dynamics, efficiency and stability in both bioreactors were examined at different dilution rates ( D = 0.0333–0.1667 h −1). In both cases, bioprocess dynamics was monitored by the analysis of biomass, substrate and metabolites concentrations in the liqiud outflow. The bioprocess efficiency and stability was much higher in HRTB than in CSTR. Advantages of HRTB over CSTR were more obvious at higher dilution rates as a consequence of bacterial biofilm formation on the inner surface of HRTB that enabled better biomass retention.

D-Galactose induces cellulase gene expression in Hypocrea jecorina at low growth rates

Lactose (1,4-O-beta-d-galactopyranosyl-d-glucose) is a soluble and economic carbon source for the industrial production of cellulases or recombinant proteins by Hypocrea jecorina (anamorph Trichoderma reesei). The mechanism by which lactose induces cellulase formation is not understood. Recent data showed that the galactokinase step is essential for cellulase induction by lactose, but growth on d-galactose alone does not induce cellulases. Consequently, the hypothesis was tested that d-galactose may be an inducer only at a low growth rate, which is typically observed when growing on lactose. Carbon-limited chemostat cultivations of H. jecorina were therefore performed at different dilution rates with d-galactose, lactose, galactitol and d-glucose. Cellulase gene expression was monitored by using a strain carrying a fusion between the cbh2 (encoding cellobiohydrolase 2, Cel6A) promoter region and the Aspergillus niger glucose oxidase gene and by identification of the two major cellobiohydrolases Cel7A and Cel6A. The results show that d-galactose indeed induces cbh2 gene transcription and leads to Cel7A and Cel6A accumulation at a low (D=0.015 h(-1)) but not at higher dilution rates. At the same dilution rate, growth on d-glucose did not lead to cbh2 promoter activation or Cel6A formation but a basal level, lower than that observed on d-galactose, was detected for the carbon-catabolite-derepressible Cel7A. Lactose induced significantly higher cellulase levels at 0.015 h(-1) than d-galactose and induced cellulases even at growth rates up to 0.042 h(-1). Results of chemostats with an equimolar mixture of d-galactose and d-glucose essentially mimicked the behaviour on d-galactose alone, whereas an equimolar mixture of d-galactose and galactitol, the first intermediate of a recently described second pathway of d-galactose catabolism, led to cellulase induction at D=0.030 h(-1). It is concluded that d-galactose indeed induces cellulases at low growth rate and that the operation of the alternative pathway further increases this induction. However, under those conditions lactose is still a superior inducer for which the mechanism remains to be clarified.

Effect of furfural on carbon metabolism key enzymes of lactose-assimilating yeasts

The metabolic response of lactose-assimilating yeasts to changes of cultivation conditions after addition of furfural into the medium was explored. Two yeast strains were studied— Candida blankii 35 with an oxidative metabolism, and C. pseudotropicalis 11 with a fermentative metabolism. Strains were cultivated in a chemostat under carbon limitation and dilution rates D = 0.1 and 0.25 h −1. During the transition period after a shock treatment with 0.04% furfural, changes of the following enzymes of the carbon metabolism were studied: hexokinase (HK), glucosephosphate isomerase (GPI) and glyceraldehyde-3P-dehydrogenase (GAPD) of the glycolysis, glucose-6P-dehydrogenase (GPD) from the pentosophosphate cycle, as well as β-galactosidase. Results show that during cultivation of C. pseudotropicalis 11 at D = 0.1 h −1, furfural concentration of 0.04% caused increase of the glycolytic enzyme activities, while the level of GPD activity remained rather constant. At the higher dilution rate D = 0.25 h −1 the effect of furfural brought to a significant decrease of all enzyme activities studied, except β-galactosidase, for which activity registered at both dilution rates was significantly higher. During cultivation of C. blankii 35 at D = 0.1 h −1, furfural brought to lower activities of GPI and GPD, while HK activity was not affected by the inhibitor, and that of GAPD increased. The furfural shock at D = 0.25 h −1 leads to increased activity of the three glycolytic enzymes, while that of GPD was 33.6% lower as compared to control. Addition of furfural into the cultivation medium of the oxidative strain caused decrease of β-galactosidase activity.

Feed intake, forestomach fluid volume, dilution rate and mean retention of fluid in the forestomach during water deprivation and rehydration in camels ( Camelus sp.)

Camels were deprived of water for 11 days. Before and during water deprivation and during rehydration changes in body weight, feed and water intake were measured. Using the liquid marker Cr-EDTA forestomach fluid volume, mean fluid retention and fluid dilution in the forestomach were estimated. At the eleventh day of water deprivation hay intake had decreased to only 9.6% of controls, dilution rates had decreased to 31%, mean retention time of fluid in the forestomach had increased to 189%. At the end of dehydration flow of saliva of 2 l/h mainly contributed to the still rather high dilution rates. Thereby buffering capacity and flow of fluid into the forestomach for microbial digestion as well as the outflow from the forestomach were maintained. At the beginning of rehydration camels drank 97 l within a few minutes, and animals thereby replaced all the water lost. Following this first huge water intake water is rapidly absorbed from the forestomach, and forestomach volume decreased again to dehydration values. At the third day of rehydration control values were reached again. Although feed intake decreased dramatically during water deprivation, functions of the forestomach can be maintained sufficiently mainly due to saliva inflow. This explains the mostly rapid recovery of camels when water is available again.

Microbial community of a mesophilic propionate-degrading methanogenic consortium in chemostat cultivation analyzed based on 16S rRNA and acetate kinase genes

We constructed a mesophilic anaerobic chemostat that was continuously fed with synthetic wastewater containing propionate as the sole source of carbon and energy. Steady-state conditions were achieved below the critical dilution rate of 0.3 d (-1) with almost complete substrate degradation. The propionate-degrading methanogenic communities in the chemostat at dilution rates of 0.01, 0.08, and 0.3 d (-1) were analyzed using molecular biological techniques. Fluorescence in situ hybridization with archaeal and bacterial domain-specific probes showed that archaeal cells predominated throughout the three dilution rates. Archaeal-16S rRNA gene clone library analysis and quantitative real-time polymerase chain reaction studies showed that hydrogenotrophic methanogen rRNA genes closely related to Methanoculleus was detected at a dilution rate of 0.01 d (-1) , whereas rRNA genes closely related to the Methanoculleus and Methanospirillum genera were detected at dilution rates of 0.08 and 0.3 d (-1) . The aceticlastic methanogen, Methanosaeta , was detected throughout the three dilution rates. Bacterial-rRNA gene clone library analysis and denaturing gradient gel electrophoresis demonstrated that rRNA genes affiliated with the genus Syntrophobacter predominated at the low dilution rate, whereas rRNA genes affiliated with the phylum Firmicutes predominated at the higher dilution rates. A significant number of rRNA genes affiliated with the genus Pelotomaculum were detected at dilution rate of 0.3 d (-1) . The diversity of genes encoding acetate kinase agreed closely with the results of the rRNA gene analysis. The dilution rates significantly altered the archaeal and bacterial communities in the propionate-fed chemostat.

Growth rate and nutrient limitation affect the transport of Rhodococcus sp. strain DN22 through sand

Rhodococcus strain DN22 grows on the nitramine explosive RDX as a sole nitrogen source, and is potentially useful for bioremediation of explosives-contaminated soil. In order for strain DN22 to be effectively applied in situ, inoculum cells must reach zones of RDX contamination via passive transport, a process that is difficult to predict at field-scale. We examined the effect of growth conditions on the transport of DN22 cells through sand columns, using chemostat-grown cultures. Strain DN22 formed smaller coccoid cells at low dilution rate (0.02 h(-1)) and larger rods at high dilution rate (0.1 h(-1)). Under all nutrient limitation conditions studied, smaller cells grown at low dilution rate were retained more strongly by sand columns than larger cells grown at high dilution rate. At a dilution rate of 0.05, cells from nitrate-limited cultures were retained more strongly than cells from RDX-limited or succinate-limited cultures. Breakthrough concentrations (C/C (0)) from sand columns ranged from 0.04 (nitrate-limited, D=0.02 h(-1)) to 0.98 (succinate-limited, D=0.1 h(-1)). The observed strong effect of culture conditions on transport of DN22 cells emphasizes the importance of physiology studies in guiding the development of bioremediation technologies.

Enhanced and potential degradation of o-phthalate by Bacillus sp. immobilized cells in alginate and polyurethane

Phthalate is a key metabolite during the degradative pathways of various aromatic compounds, which are generally recognized as pollutants. Bacillus sp. (NCIM 5220), a soil microbe, is capable of utilizing o-phthalate as the sole carbon and energy source. This bacterium was immobilized in alginate and polyurethane foam (PUF). The degradation of phthalate by free cells was compared with the degradation by alginate- and PUF-immobilized cells in shaken batch, repeated batch, and continuous degradation in a packed bed reactor. The immobilized cells in alginate and PUF showed higher degradation of phthalate than free cells and could be used for repeated cycles for more than 12 and 24 cycles, respectively. Further the immobilized cells could be operated for the degradation of phthalate in a packed-bed reactor with much higher dilution rates. Thus, more efficient degradation of phthalate was achieved by immobilizing the Bacillus sp. in alginate and polyurethane foam.

Growth dynamics of Azospirillum lipoferum at steady and transitory states in the presence of NH

The objective of this paper was to study the adaptation dynamics and biochemical response of Azospirillum lipoferum grown in a continuous culture at various environmental shifts. The kinetics of A. lipoferum Sp 59b grown at steady states in a microaerobic chemostatic environment deviated from a typical Monod kinetics in both low and high dilution rates (D) due to several metabolic shifts that occurred in the microbial cell. When NH4Cl was exhausted (at low D), the microbial cell partitioned carbon flow in order to sustain growth, nitrogen fixation and assimilation processes (occurred via the glutamate synthase reaction). Increasing D the specific activities of the enzymes involved in the tricarboxylic acid cycle and the respiration rate were decreased. At transitory states, under optimal for nitrogen fixation dissolved oxygen (DO) concentrations, ammonium nitrogen negatively affected, besides nitrogen fixing activity, the bacterial growth. At sub-optimal for nitrogen fixation DO concentration (i.e. 1.56 microM) and 0.1 g l(-1) NH4Cl in the fed medium, the activities of citrate synthase and succinate dehydrogenase were significantly reduced. Important shifts in both carbon and nitrogen metabolism occur in A. lipoferum grown in the presence of the ammonium nitrogen, while the boundaries of ammonium nitrogen concentration in which A. lipoferum can be adapted depend on the DO concentration in the growth environment. Studies on growth dynamics and physiology of A. lipoferum, grown in experimental model systems, can contribute to an efficient application of these bacteria as plant-growth-promoting-agents.

The role of NADH-oxidation in acetoin and diacetyl production from glucose in Lactococcus lactis subsp. lactis MG1363

Product formation from glucose and NADH-oxidase activity from Lactococcus lactis MG1363, a non-citrate fermenter, was studied under aerobic conditions. In continuous cultures constantly aerated to 80% air saturation, the usual homolactic fermentation was almost completely replaced by acetate fermentation (80–90% of the fermented glucose) at low dilution rates. As the dilution rate was increased, the homolactic fermentation was partially restored, however the glucose fermentation profile was pH dependent. Significant production of acetoin and diacetyl was found at low pH and high dilution rates. NADH-oxidase activity was studied under different culture conditions. An up to fivefold increase in NADH-oxidase activity was observed in batch culture when air tension was increased from 0% to 80%. In aerobic continuous cultures at 80% air saturation, the NADH-oxidase activity was directly dependent on dilution rate and growth pH. The observed variations in NADH-oxidase activity played a crucial role in pyruvate flux redistribution and clearly correlated with the observed shifts in end product formation from glucose.