Dissolved oxygen concentration-controlled feeding of substrate into Kluyveromyces fragilis culture

b-Galactosidase production by Kluyveromyces fragilis was optimized by controling dissolved oxygen concentration (DOC) due to the enzyme production rate being strongly dependent on this parameter in the broth. A system of automatic control of substrate inflow which depends on DOC has been devised. This strategy has resulted in a significant and reproducible improvement in the performance of the fermentation. Volumetric enzyme production rates were greater in this system than in batch culture of the microorganism with DOC control.     

Interaction of <Emphasis Type="Italic">Klebsiella oxytoca</Emphasis> and <Emphasis Type="Italic">Burkholderia cepacia</Emphasis> in Dual-Species Batch Cultures and Biofilms as a Function of Growth Rate and Substrate Concentration

Dual-species microbial interactions have been extensively reported for batch and continuous culture environments. However, little research has been performed on dual-species interaction in a biofilm. This research examined the effects of growth rate and substrate concentration on dual-species population densities in batch and biofilm reactors. In addition, the feasibility of using batch reactor kinetics to describe dual-species biofilm interactions was explored. The scope of the research was directed toward creating a dual-species biofilm for the biodegradation of trichloroethylene, but the findings are a significant contribution to the study of dual-species interactions in general. The two bacterial species used were Burkholderia cepacia PR1-pTOM_31c, an aerobic organism capable of constitutively mineralizing trichloroethylene (TCE), and Klebsiella oxytoca, a highly mucoid, facultative anaerobic organism. The substrate concentrations used were different dilutions of a nutrient-rich medium resulting in dissolved organic carbon (DOC) concentrations on the order of 30, 70, and 700 mg/L. Presented herein are single- and dual-species population densities and growth rates for these two organisms grown in batch and continuous-flow biofilm reactors. In batch reactors, planktonic growth rates predicted dual-species planktonic species dominance, with the faster-growing organism (K. oxytoca) outcompeting the slower-growing organism (B. cepacia). In a dual-species biofilm, however, dual-species planktonic growth rates did not predict which organism would have the higher dual-species biofilm population density. The relative fraction of each organism in a dual-species biofilm did correlate with substrate concentration, with B. cepacia having a greater proportional density in the dual-species culture with K. oxytoca at low (30 and 70 mg/L DOC) substrate concentrations and K. oxytoca having a greater dual-species population density at a high (700 mg/L DOC) substrate concentration. Results from this research demonstrate the effectiveness of using substrate concentration to control population density in this dual-species biofilm.     

Improved production of citric acid by Yarrowia lipolytica using oleic acid as the oxygen‐vector and co‐substrate

Yarrowia lipolytica is able to secrete large amounts of citric acid (CA), which is greatly affected by the dissolved oxygen concentration (DOC) in the fermentation medium. In this study, oleic acid was selected as oxygen‐vector to improve DOC during CA fermentation. When 2% (v/v) of oleic acid was added to the culture broth, higher DOC (>42.1%) was determined throughout the CA synthesis phase. The yield of CA reached a maximum of 32.1 g/L (25.4% higher than the control) and the biomass was 8.8 g/L. The substrate uptake rate, products formation rate and key enzyme activities were also determined, and the results indicated that CA synthesis was strengthened with oleic acid addition. Furthermore, it was detected that oleic acid could be assimilated by the cells, which means that oleic acid could be served both as oxygen‐vector and co‐substrate for CA synthesis by Y. lipolytica. In a bioreactor with working volume of 3 L, the highest concentration of CA reached to 36. 4 g/L in the presence of 2% (v/v) oleic acid after 192 h of fermentation. These results confirmed that oleic acid could be applied in the large‐scale production of CA by Y. lipolytica.     

Fermentation of n-Paraffins by Yeast

Screening test for obtaining microorganisms which produce l-amino acids or organic acids from n-paraffins were carried out. Fourteen strains of microorganisms which seemed to belong to the yeast showed strong ability to produce α-ketoglutaric acid. A representative strain of these microorganisms was identified as Candida lipolytica AJ 5004.

Optimal conditions for production of α-ketoglutarate using Candida lipolytica AJ 5004 were also studied. Under the condition thus obtained using a culture medium of 8 weight % of n-paraffins, the yeast accumulated 59% of α-ketoglutarate to the substrate added after three days culture.     

Model of dissolved organic carbon distribution for substrate-sufficient continuous culture.

The growth yields (Yobs) are greater under substrate-limited conditions than those under substrate-sufficient conditions in continuous cultures. This indicates that the excess substrate should cause uncoupling between anabolism and catabolism. It appears that the excess substrate could determine metabolic pathways of microorganisms, which further control dissolved organic carbon (DOC) distribution under substrate-sufficient conditions. However, how to quantitatively describe the DOC distribution remains unclear in substrate-sufficient continuous culture. Based on a balanced DOC reaction, a DOC distribution model was developed in relation to residual substrate concentration for substrate-sufficient continuous cultures. Results showed that a considerable portion of the DOC consumed was directly oxidized to carbon dioxide through energy spilling under substrate-sufficient conditions. The proposed model for the first time quantified the DOC distribution between nongrowth-associated and growth-associated metabolisms of cells. The proposed model was verified with literature data very well.     

Impact of balanced substrate flux on the metabolic process employing fuzzy logic during the cultivation of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> var.<Emphasis Type="Italic"> Galleriae</Emphasis>

Summary The insecticidal crystal protein (ICP) synthesized at the onset of sporulation by Bacillus thuringiensis var. galleriae (Btg) is lethal against specific pests. Attempts were made to enhance the synthesis of biomass and ICP by Btg employing process optimization strategies. The process optimization was carried out with residual glucose concentration control in a bench scale bioreactor. A fuzzy logic-based feedback control system for maintaining the residual glucose concentration at a constant level during cultivation was developed in LabVIEW. This control system indicated the possibilities in providing a balanced substrate flux during cultivation. The identified optimum level of 2.72 g/l in residual glucose concentration was maintained by fed-batch cultivation with glucose and yeast extract fed at equal concentration with the above control system. High cell density of 16.0 g/l with specific growth rate of 0.69 h^-1 was obtained during the cultivation. The balanced flux of substrate during cultivation has resulted in the enhanced synthesis of biomass and ICP. This optimized process could be commercially exploited by comparing the fluxes of basal compounds in different media sources used in fermentation.     

Effect of organophosphorus insecticides on the oxidative processes in rat brain synaptosomes

Abstract: This paper describes the effect of four organophosphorus insecticides: Dipterex, DDVP, Ronnel and its oxygen analogue on the respiration of rat brain synaptosomes. Dipterex and DDVP in the concentrations used, 5, 50, or 500 μM, did not change the rate of oxygen uptake and oxidative phosphorylation in rat brain synaptosomes. Ronnel in the highest concentration (500 μM) inhibited respiration in state 3 conditions and abolished respiratory control by ADP. This inhibition was correlated with a change of cytochrome c oxidase activity. The oxygen analogue of Ronnel (OAR) in micromolar concentrations (50 μM) increased the rate of respiration of synaptosomes utilizing glutamate plus malate as substrate. Higher concentrations of OAR produced inhibition of respiration, cytochrome c oxidase and NADH: cytochrome c reductase activities. These observations are typical for uncouplers of oxidative phosphorylation. Noteworthy is the fact that the uncoupling activity of OAR was observed at concentrations which did not inhibit acetylcholinesterase activity. These findings seem to suggest that disturbances in oxidative processes could play an important role in the toxicity of organophosphorus insecticides. The relation between chemical structure and the ability of insecticides to affect oxidative phosphorylation is discussed.     

Der substratlimitierte pH-Auxostat – Eine neue Methode zur kontinuierlichen Kultur

A method for continuous cultivation of microorganisms is demonstrated, the substrate limited pH-auxostat. The limiting substrate only is added with constant velocity. In this culture the cells grow with high utilization of the limiting substrate and with the highest specific growth rate possible at the given conditions. Yield coefficients and dilution rates of stable K⁺-limited steady states in yeast cultures with different pH-values and biomass concentrations were measured.     

Fermentative bioconversion in a horizontal rotating tubular bioreactor

The performance of a horizontal rotating tubular bioreactor (HRTB) was investigated with a biological system under non-sterile conditions. A spontaneously developed microbial culture was cultivated in a simple glucose/yeast extract medium. A fermentative bioconversion was examined by different combinations of process parameters (bioreactor rotation speed 5–30 min⁻¹ and medium inflow rate 1–10 l h⁻¹). Bioconversion dynamics in HRTB was monitored by withdrawing the samples from five positions along the bioreactor. Investigation in HRTB showed a rapid and an efficient glucose conversion into different products of metabolism. Glucose consumption rate along the HRTB depended on medium inflow rate, while bioreactor rotation speed did not have a significant influence. Complete glucose conversion in HRTB was observed at inflow rates of up to 6.5 l h⁻¹. The pH gradient along the HRTB was detected at higher medium inflow rates (6.5 and 10 l h⁻¹), but did not significantly influence substrate conversion efficiency. A discussion of its potential use and a comparison of HRTB with other bioreactors are also presented.     

A kinetic model incorporating energy spilling for substrate removal in substrate-sufficient batch culture of activated sludge

Batch assays are currently used to study the kinetic behavior of microbial growth. However, it has been shown that the outcome of batch experiments is greatly influenced by the initial ratio of substrate concentration (S _o) to biomass concentration (X _o). Substrate-sufficient batch culture is known to have mechanisms of spilling energy that lead to significant nongrowth-associated substrate consumption, and the Monod equation is no longer appropriate. By incorporating substrate consumption associated with energy spilling into the balance of the substrate oxidation reaction, a kinetic model for the observed specific substrate consumption rate was developed for substrate-sufficient batch culture of activated sludge, and was further verified by experimental data. It was demonstrated that the specific substrate consumption rate increased with the increase of the S _o/X _o ratio, and the majority of substrate was consumed through energy spilling at high S _o/X _o ratios. It appears that the S _o/X _o ratio is a key parameter in regulating metabolic pathways of microorganisms. Received: 18 January 1999 / Received revision: 7 May 1999 / Accepted: 28 May 1999     

Dynamics and control of continuous microbial propagators to subject substrate inhibition

Inhibitory substrate levels are common in industrial fermentations and in biological waste-water treatment of many industrial wastes. Continuous microbial cultures are unstable to certain disturbances, such as shock loading by inhibitory substrates. Two feedback proportional control strategies are analyzed and compared for a simple model culture assumed represent able by the culture concentrations of biomass and a single rate-limiting and growth-limiting nutrient (substrate). One control strategy, the well known turbidostat, consists of adjusting culture holding time (e.g., by flow rate adjustment) in response to deviations in turbidity or some other measure of culture biomass concentration. The other control strategy is to adjust holding time in response to deviations in limiting nutrient concentrations in the culture. This second control strategy, termed the nutristat, can be superior to the turbidostat in many applications. The sign and magnitude of the dimensionless group {(X /YD )[dμ/dS]s }, is shown to be an important determinant, in the behavior of the open loop and the two closed loop processes. This characteristic group is positive when the specific growth rate is increased by increases in the nutrient concentration, zero when the growth rate is unaffected by the nutrient concentration, and negative in the presence of nutrient or substrate inhibition. The effects of process modifications and of modeling assumptions on the control of the process are discussed and more sophisticated control schemes are also proposed.     

降解剂对森林凋落物可溶性有机碳含量的影响及凋落物降解模式比较研究

森林凋落物是森林生态系统中可溶性有机碳(DOC)的主要来源,在生态系统碳循环过程中起重要作用。以帽儿山地区胡桃楸、兴安落叶松以及胡桃楸-兴安落叶松人工林凋落物为研究对象,通过液体发酵培养将纤维素高效降解真菌Peniophora intranata与Sarocladium strictum制成单一(A菌剂、B菌剂)及混合菌剂(C菌剂),测定野外条件下经降解剂处理的凋落物基质在不同时期的DOC含量,分析降解剂对不同类型森林凋落物DOC动态变化的影响,并比较各凋落物基质降解模式异同。结果表明：(1)凋落物基质DOC含量在降解剂处理后均随降解时间增加呈下降趋势,且下降幅度大小表现为混合基质>胡桃楸基质>落叶松基质;在最初的1个月,各凋落物基质DOC含量均显著高于其他降解时期DOC含量。(2)经混合菌剂处理后的胡桃楸基质DOC含量相较于2种单一菌剂处理后的胡桃楸基质DOC含量低,而经混合菌剂处理后的兴安落叶松基质与胡桃楸-兴安落叶松基质的DOC含量未表现出相同的显著性。(3)经3种菌剂处理后的胡桃楸基质降解模式均相同,经B菌剂与C菌剂处理的落叶松基质降解模式相同,经A菌剂与C菌剂处理...     

Substrate inhibition kinetics of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> in fed-batch cultures operated at constant glucose and maltose concentration levels

Fed-batch culture is the mode of operation of choice in industrial baker’s yeast fermentation. The particular mode of culture, operated at stable glucose and maltose concentration levels, was employed in this work in order to estimate important kinetic parameters in a process mostly described in the literature as batch or continuous culture. This way, the effects of a continuously falling sugar level during a batch process were avoided and therefore the effects of various (stable) sugar levels on growth kinetics were evaluated. Comparing the kinetics of growth and the inhibition by the substrate in cultures grown on glucose, which is the preferential sugar source for Saccharomyces cerevisiae, and maltose, the most common sugar source in industrial media for baker’s yeast production, a milder inhibition effect by the substrate in maltose-grown cells was observed, as well as a higher yield coefficient. The observed sugar inhibition effect in glucostat cultures was taken into account in modeling substrate inhibition kinetics. The inhibition coefficient K _i increased with increasing sugar concentration levels, but it appeared to be unaffected by the type of substrate and almost equal for both substrates at elevated concentration levels.     

海水中藻菌共培养体系对碳氮磷的吸收转化

海洋环境中,细菌和微藻之间的物质交换是生源要素在自然界中迁移转化的重要方式。为进一步了解生源要素的生物地球化学循环,在实验室模拟条件下,研究了共培养体系中营养盐和有机物在细菌和微藻之间的转换。通过纯培养中肋骨条藻(Skeletonema costatum)、东海原甲藻(Prorocentrum donghaiense)、天然海水中的细菌以及藻菌混合培养,分析了营养盐和有机物随藻菌生物量的变化情况,并计算了溶解有机碳(DOC)和溶解有机氮(DON)的浓度比值[(DOC/DON)a]。结果发现,在共培养体系中,细菌对中肋骨条藻的生长有抑制作用,对东海原甲藻影响不明显;中肋骨条藻有利于细菌生长,东海原甲藻抑制细菌生长,这种不同可能与微藻的粒径有关。海洋细菌在2种藻的指数生长均期均会促进微藻吸收氨氮(NH_4-N),但在生长末期NH_4-N以释放为主。硝氮(NO_3-N)的浓度与藻的生长呈负相关,但在衰亡期NO_3-N略有增加,表明NO_3-N再生所需时间较长。细菌对硝氮的吸收量较少,但对其再生有贡献。细菌和中肋骨条藻对磷酸盐(PO_4-P)的吸收存在竞争,但与东海原甲藻的竞争关系不明显。不同培养体系中DOC浓度变化不同,在藻菌共培养体系中增加较快,纯藻培养体系中增加缓慢,在纯菌培养体系中缓慢减少。通过对DOC与DON浓度比值的分析,发现用判断颗粒有机碳(POC)来源的方法可以分析DOC的来源。     

A new dynamic model for highly efficient mass transfer in aerated bioreactors and consequences for kLa identification

Gas–liquid mass transfer is often rate‐limiting in laboratory and industrial cultures of aerobic or autotrophic organisms. The volumetric mass transfer coefficient k_La is a crucial characteristic for comparing, optimizing, and upscaling mass transfer efficiency of bioreactors. Reliable dynamic models and resulting methods for parameter identification are needed for quantitative modeling of microbial growth dynamics. We describe a laboratory‐scale stirred tank reactor (STR) with a highly efficient aeration system (k_La ≈ 570 h^?1). The reactor can sustain yeast culture with high cell density and high oxygen uptake rate, leading to a significant drop in gas concentration from inflow to outflow (by 21%). Standard models fail to predict the observed mass transfer dynamics and to identify k_La correctly. In order to capture the concentration gradient in the gas phase, we refine a standard ordinary differential equation (ODE) model and obtain a system of partial integro‐differential equations (PIDE), for which we derive an approximate analytical solution. Specific reactor configurations, in particular a relatively short bubble residence time, allow a quasi steady‐state approximation of the PIDE system by a simpler ODE model which still accounts for the concentration gradient. Moreover, we perform an appropriate scaling of all variables and parameters. In particular, we introduce the dimensionless overall efficiency κ, which is more informative than k_La since it combines the effects of gas inflow, exchange, and solution. Current standard models of mass transfer in laboratory‐scale aerated STRs neglect the gradient in the gas concentration, which arises from highly efficient bubbling systems and high cellular exchange rates. The resulting error in the identification of κ (and hence k_La) increases dramatically with increasing mass transfer efficiency. Notably, the error differs between cell‐free and culture‐based methods of parameter identification, potentially confounding the determination of the “biological enhancement” of mass transfer. Our new model provides an improved theoretical framework that can be readily applied to aerated bioreactors in research and biotechnology. Biotechnol. Bioeng. 2012; 109: 2997–3006. © 2012 Wiley Periodicals, Inc.     

Regulation of the biosynthesis of cytochrome P-450 in brewer's yeast role of cyclic AMP

The drug metabolising enzyme cytochrome P-450 has been studied in great detail in mammalian systems and its presence in microorganisms is also well established. However, neither its function nor its means of control in brewer's yeast, Saccharomyces cerevisiae, has been investigated. We demonstrate here using yeast protoplasts that it is the intracellular concentration of cyclic AMP which controls, by repression, the de novo synthesis of the enzyme, and also that cyclic AMP concentrations are in turn inversely related to the concentration of glucose in the yeast growth medium.     

Inferential control of the specific growth rate in fed-batch cultivation processes

Inferential control algorithms and systems for set-point control of the specific growth rate in fed-batch cultivation processes are presented. Realization of the proposed control systems does not require mathematical model and a priori knowledge of the culture of microorganisms under control. Feed-back signal in PID control loops of the investigated systems is based on measurements of the O₂ concentration in exhaust gas and the air supply rate, however, measurements of the other technological parameters related to the culture growth dynamics can also be applied.     

The production of ethanol by immobilized yeast cells

Saccharomyces cerevisiae cells were immobilized in calcium alginate beads for use in the continuous production of ethanol. Yeasts were grown in medium supplemented with ethanol to selectively screen for a culture which showed the greatest tolerance to ethanol inhibition. Yeast beads were produced from a yeast slurry containing 1.5% alginate (w/v) which was added as drops to 0.05M CaCl₂ solution. To determine their optimum fermentation parameters, ethanol production using glucose as a substrate was monitored in batch systems at varying physiological conditions (temperature, pH, ethanol concentration), cell densities, and gel concentration. The data obtained were compared to optimum free cell ethanol fermentation parameters. The immobilized yeast cells examined in a packed-bed reactor system operated under optimized parameters derived from batch-immobilized yeast cell experiments. Ethanol production rates, as well as residual sugar concentration were monitored at different feedstock flow rates.     

Relative importance of inefficient feeding and consumer excretion to organic carbon flux from Daphnia

1. Sloppy and inefficient feeding by zooplankton is generally thought to make a major contribution to the regeneration of the dissolved organic carbon (DOC) pool in aquatic environments. In this study, we tested experimentally the regeneration of DOC by a freshwater zooplankter feeding on two species of phytoplankton at different food concentrations and C : P ratios. We separated the DOC production because of inefficient feeding (pre‐ingestive regeneration) and zooplankton excretion and faeces release (postdigestive regeneration). 2. Within a brief incubation period (10 min), DOC production in the presence of Daphnia was not significantly different from that in the control treatment without grazers. During a longer incubation period (4 h), the amounts of radiocarbon retained in the algal cells per se were constant or were not different from those in the control treatments. These experimental results strongly suggest that inefficient feeding did not contribute significantly to DOC production in the grazer–prey system. 3. During the 4‐h incubation, calculations of the DOC per ingestion rate (i.e. DOC produced by Daphnia alone) showed that food concentration and algal species did not affect the relative DOC production, but there was considerable difference at different algal C : P ratios and grazer densities. We found that direct excretion of DOC by Daphnia occurred rapidly following food digestion and accounted for >65% of the total DOC production. Maximum DOC leakage from Daphnia faeces contributed less to DOC production than the grazer excretion, except under P‐limited conditions. 4. This study highlights the dominant role of postingestive process, especially the direct excretion by zooplankton, in DOC production in a grazer–prey system.