Significance of inoculum size in biofilm formation by staphylococci

In the present study the effect of inoculum size, ranging from 10(6) to 1 cell, in biofilm formation by staphylococci was determined by microtiter plate test. The initial inoculum size had a dramatic effect on the quantity of biofilm formed. A decreased number of bacteria in initial inoculum always resulted in a decreased production of biofilm.     

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.     

Investigation of parameters affecting a fixed bed bioreactor process for recombinant cell lines

A BHK 21 cell line expressing a recombinant antibody was grown in a fixed bed reactor (FBR) system using a porous support made of Siran glass beads. The contribution of five process variables (bead and inoculum sizes; circulation and dilution rates; glutamine concentration of the feed) to the productivity of the process (defined as production rate, effluent product concentration or yield of product on medium supplied) was investigated using a partial factorial experimental design. Individually, none of the variables tested had a significant affect upon productivity. The combination of smaller bead and inoculum sizes, higher circulation and dilution rates, plus higher feed glutamine concentration gave a markedly higher productivity than any other combination of variable levels tested. This combination of variable levels suggested that better results shold be obtained using a fluidised bed reactor system. However, comparison of the productivities of the two systems showed that the FBR gave the better results. This result can be explained in terms of the relationship of Qs_rAb to .Abbreviations C concentration - D dilution rate - FBR fixed bed bioreactor - FIBR fluidised bed bioreactor - Gln glutamine - Qs cell specific rate - Qv volumetric rate - rAb recombinant antibody - X_v viable cell density - specific growth rate     

Role of anaerobic spore-forming bacteria in the acidogenesis of glucose: Changes induced by discontinuous or low-rate feed supply

A mineral salts medium containing 1% (w/v) glucose providing carbon-limited growth conditions was subjected to anaerobic acidogenesis by mixed populations of bacteria in chemostat cultures. The formation of butyrate was shown to be dependent on the presence of saccharolytic anaerobic sporeformers in the acid-forming population. By the use of pasteurized activated sludge as an inoculum a culture was obtained consisting solely of anaerobic sporeformers that gave rise to the formation of butyrate, acetate, hydrogen and carbon dioxide as the main fermentation products. No formation of propionate could be detected. In this culture, the role of sporulation was investigated by applying periods of starvation and a single-step lowering of dilution rate (shift-down). In an experiment using a mineral salts medium supplemented with 1% (w/v) glucose and 0.5% (w/v) casein hydrolysate formation of refractile forespores as well as cell lysis could be demonstrated after 6 h starvation.In mixed cultures, initially inoculated with non-pasteurized activated sludge, a regular interruption of feed supply for 1 h per day resulted in selection of non-sporulatiog anaerobes. The fermentation pattern changed to a production of propionate and acetate, with a concomitant reduction of gas production. Similar results were obtained with shift-down in dilution rate.     

Mycelial morphology and fungal protein production from starch processing wastewater in submerged cultures of Aspergillus oryzae

Three strains of Aspergillus oryzae were used for fungal protein production from starch processing wastewater (SPW) as part of a comprehensive SPW utilization and treatment program. The mycelial morphologies of the three strains varied from fluffy, clumpy mycelia to compact pellets with various strains and cultivation conditions of growth pH, inoculum quantity and superficial air velocity in an air lift bioreactor process. An inoculum technique was developed to ensure a desirable morphology with an improved fungal protein yield. Experimental relationship between the morphology and the yield revealed that the formation of small compact pellets under designed cultivation conditions favoured higher fungal protein yields, easier product separation and better process operation.     

Mathematical Model and Mechanisms for Biofilm Wastewater Treatment Systems

Summary Fundamental theoretical depiction is still lacking on biofilm wastewater treatment systems up to today. A mathematical model of biofilm wastewater treatment systems, taking account of suspended microorganisms and some factors influencing biofilm formation and stabilization, is developed in this paper. By theoretical and numerical analyses, the factors influencing biofilm formation and stabilization, such as the dilution rate, influent organic concentration, detachment and initial inoculum concentration etc, are discussed. Qualitative investigations were carried out and suggestions on industrial applications are then proposed. This paper not only plays an important role in understanding the physical mechanisms of biofilm dynamics, but also has far-reaching implications for industrial practices.     

Changes of respiration and of specific growth rate during cell cycle of yeast cells of different genealogical age

When investigating changes of respiratory activity during the cell cycle of mother and daughterCandida cells significant oscillations of specific rate of oxygen consumption were detected; specific growth rate also varied. The oscillations were less pronounced when the inoculum was obtained from the chemostat at the high dilution rates of 0.25 and 0.35/h.     

UASB反应器中影响污泥颗粒化的工程因素

研究了具有不同微生物群系的接种污泥、流动方式和流速对上流式厌氧污泥床(UASB)反应器中活性污泥粒化的影响。颗粒化过程包括：微生物絮凝体的形成、亚核的形成,亚核增长和颗粒成熟四个阶段。微絮凝体的形成取决于酸化菌的作用。流体的动量传递和流体对悬浮物的剪切作用是影响亚核形成的关键性工程因素。为此提出最低流速概念,即形成污泥膨胀床的最低流速。合适的进料速率、污泥负荷、布水均匀性以及碱度控制是UASB反应器工程放大和过程控制的四大要素。     

The effect of incubation conditions on the laboratory measurement of the methane producing capacity of livestock wastes

The effects of incubations conditions (dilution, mixing, incubation time and inoculum amount and origin) on the determination of the maximum methane producing capacity (B(0)) from various livestock slurries were evaluated. For this purpose, the methane yields of different livestock slurries were determined using batch anaerobic incubations performed at 30 degrees C as regard these different conditions. The B(0) and the methane (CH(4)) generation as a function of time were used to study the processes and to determine the best incubation conditions. Methanogenesis was identified as the major rate-limiting step during the anaerobic degradation of slurries, probably due to inhibition by volatile fatty acids. In some cases, high free NH(3) concentrations were suspected to inhibit the hydrolysis process. The addition of inoculum and/or the dilution of the substrate reduced the inhibition and allowed to reach the B(0) more rapidly. However, the addition of inoculum must be minimized to reduce the possible errors made by considering a similar production by the inoculum with and without the substrate. All experiments performed during this study allowed to define the incubation conditions required for the determination of the B(0) from livestock slurries. Applying these conditions, the B(0) values determined for swine slurries varied from 244 to 343L CH(4)kg V(added)(-1), from 204 to 296L CH(4)kg V(added)(-1) for dairy cattle slurries and equalled 386 and 319L CH(4)kg VS(added)(-1) respectively for calves and duck slurries.     

Physiological factors affecting streptomycin production by Streptomyces griseus ATCC 12475 in batch and continuous culture

Abstract Conditions of growth are described for the production of streptomycin by Streptomyces griseus ATCC 12475 using chemically defined minimal medium and complex medium. It was found using batch cultures that early synthesis of the antibiotic occurred during growth in minimal medium but was delayed until the onset of stationary phase in complex medium. This effect was independent of whether spores or vegetative cells were used as inoculum. Stability of streptomycin biosynthesis in continuous culture was dependent on dilution rate and medium employed. Cultures were highly unstable when grown on complex medium but could be maintained in steady states in continuous culture using minimal medium when the dilution rate was increased in a stepwise manner, starting at a dilution rate of 0.02 h⁻¹ (15% of μ _max). The effect of changing dilution rate on growth, streptomycin production and the level of streptomycin phosphotransferase was examined using this technique.     

The dynamics of the microorganism count of active sludge during the aerobic treatment of sewage

Dynamics of the active sludge microorganism quantity is studied under different cultivation regimes. It is shown that quantity of microorganisms in different physiological groups depends on the specific growth rate, micro, determined by the dilution rate and biomass recirculation level. The results may be used for selecting optimal regime of the sewage treatment system functioning.     

初始条件对高浓度乙醇连续发酵过程的影响及振荡行为提高发酵效率的机理分析

前期实验在稀释速率为0.027h-1的高浓度乙醇连续发酵过程中,发现了一种长周期、宽振幅的参数振荡现象。本实验进一步考察了不同稀释速率下的连续发酵过程,发现在稀释速率为0.04h-1条件下,也能出现类似的振荡现象;在稀释速率为0.027h-1或0.04h-1的条件下,改变系统的初始状态可以得到振荡和稳态两种不同的发酵过程。比较振荡和稳态过程的实验数据后,发现在稀释速率为0.04h-1的条件下,与稳态过程相比,振荡过程的平均残糖浓度降低了14.8%,平均乙醇浓度提高了12.6%,平均设备生产强度提高了12.3%。进一步分析表明:与稳态过程相比,振荡过程动力学行为不仅存在滞后,而且在相同残糖和乙醇浓度条件下,所对应的平均比生长速率提高了53.8%。     

A continuous multistage roller reactor for animal cell culture: 1. Patterns of growth,production and catabolism of a murine hybridoma

A Tubular Liquid Film Reactor was designed as a model system to transfer a batch culture kinetic to a continuous cascade. Cell density, product formation and substrate consumption rates were followed during fermentation at two dilution rates. In spite of the high dilution rates effective in each segment by itself high cell densities of up to 10⁷ cells/ml were achieved due to cell sedimentation. The model character of the reactor was taken to determine critical values of substrate concentrations that influence production rates and result in an adaptation of metabolism.Abbreviations TLFR tubular liquid film reactor     

Application of a two-compartment model to the wall growth of Pelobacter acidigallici under continuous culture conditions

Continuous cultivation of Pelobacter acidigallici on gallic acid is accompanied by biofilm formation. Steady states can be obtained at dilution rates nearly twice the maximum growth rate. A two-compartment model is presented which allows us to determine rate constants of attachment and detachment. The model is based on the assumption that the biofilm is fully penetrated by the substrate. At high dilution rates the outcome of our experiments is mainly determined by the attachment rate. The influence of the thickness of the biofilm is discussed in order to demonstrate the limitations of the model.     

THE ROLE OF ENDOMYCORRHIZAE IN REVEGETATION PRACTICES IN THE SEMI-ARID WEST. III. VERTICAL DISTRIBUTION OF VESICULAR-ARBUSCULAR (VA) MYCORRHIZA INOCULUM POTENTIAL

The changes in vesicular-arbuscular (VA) mycorrhiza inoculum potential (MIP) in soil profiles from a mid-elevation sage community were measured using a corn bioassay. The MIP was significantly reduced below 30 cm depth and approached zero at less than 1 m depth. The decrease in inoculum potential with depth in diluted soils did not always parallel changes in the nondiluted soil, indicating factors other than numbers of inoculum units also may be important in determining the extent of mycorrhiza formation. The relationship of these results to land disturbance and associated dilution of populations of VA mycorrhizal fungi and to defining topsoil is discussed.     

The use of multi-parameter flow cytometry to study the impact of limiting substrate, agitation intensity, and dilution rate on cell aggregation during Bacillus licheniformis CCMI 1034 aerobic continuous culture fermentations

The main objective of this work was to establish those factors either physical (power input) or chemical (limiting substrate or dilution rate) that enhance cell aggregation (biofilm or floc formation) and cell physiological state during aerobic continuous cultures of Bacillus licheniformis. Glucose-limited steady-state continuous cultures growing at a dilution rate between 0.64 and 0.87/h and 1,000 rpm (mean specific energy dissipation rate (epsilonT) = 6.5 W/kg), led to the formation of a thin biofilm on the vessel wall characterized by the presence of a high proportion of healthy cells in the broth (after aggregate disruption by sonication) defined as having intact polarized cytoplasmic membranes. An increased epsilonT (from 6.5 W/kg to 38 W/kg) was found to hinder cell aggregation under carbon limitation. The carbon recovery calculated from glucose indicated that additional extracellular polymer was being produced at dilution rates >0.87/h. B. licheniformis growth under nitrogen limitation led to floc formation which increased in size with dilution rate. Counter-intuitively the flocs became more substantial with an increase in epsilonT from 6.5 W/kg to 38 W/kg under nitrogen limitation. Indeed the best culture conditions for enhanced metabolically active cell aggregate formation was under nitrogen limitation at epsilonT = 6.5 W/kg (leading to floc formation), and under carbon limitation at a dilution rate of between 0.64 and 0.87/h, at epsilonT = 6.5 W/kg (leading to vessel wall biofilm formation). This information could be used to optimize culture conditions for improved cell aggregation and hence biomass separation, during thermophilic aerobic bioremediation processes.     

Standardization of hyphal growth inhibition rate as a means of evaluating Microsporum spp. in vitro susceptibility to terbinafine, griseofulvin, and ciclopiroxolamine

Reference methods for antifungal susceptibility tests recommend the use of conidia as inoculum. However, some isolates produce few conidia, while the invasive form of filamentous fungi in general is hyphae making susceptibility tests infeaseble. These facts suggest that other than conidia broth dilution method is required for susceptibility tests. The aim of this study was to clarify if the hyphal growth inhibition rate could be used as a method of determining the antifungal susceptibility of genus Microsporum. For this reason, a method which traces hyphal tips automatically and measures their growth rate was standardized for Microsporum spp. Control growth curves and test growth curves obtained by real-time observation of the hyphae groups responses to different concentrations of terbinafine, griseofulvin, and ciclopiroxolamine were used to compare with minimum inhibitory concentrations (MICs) obtained by conidia broth microdilution method. A visible reduction in the growth inhibition rate was observed when hyphal activity was evaluated using the third or fourth serial two-fold dilution below the MIC determined by broth microdilution for terbinafine and ciclopiroxolamine. For griseofulvin, this reduction occurred after the fifth dilution below the MIC. This study highlights the importance of the inoculum type used to determine the in vitro susceptibility of Microsporum strains. We conclude that measurement of hyphal growth inhibition, despite being time consuming, could be a suitable method for evaluating antifungal susceptibility, particularly for fungi as Microsporum spp. that produce a small (or not at all) number of conidia.     

Production of extracellular protease and glucose uptake in Bacillus clausii in steady-state and transient continuous cultures

The production of the extracellular alkaline protease Savinase (EC 3.4.21.62) and glucose uptake in a non-sporulating strain of Bacillus clausii were investigated by analysing steady-state and transients during continuous cultivations. The specific production rate was found to have an optimum at a dilution rate between 0.14 and 0.17 h(-1), whereas the yield of Savinase on glucose was found to increase with decreasing specific growth rate. A linear relationship between the ribosomal RNA content and the specific production rate was found, indicating that the translational capacity may be limiting for product formation. The dynamics of the production of Savinase were studied during step changes in the dilution rate. During a step down in the dilution rate the specific production rate decreased immediately until it reached a new steady value. During a step-up an initial cease in the production rate was observed, but when glucose stopped to accumulate the production rate was regained. The glucose uptake was further investigated when chemostat cultures growing at different dilution rates were exposed to glucose pulses. The maximal glucose uptake capacity was found to be dependent on the initial specific growth rate. Furthermore, the adaptation to high glucose concentrations was faster at high dilution rates than at low dilution rates.     

Structural and Functional Responses of a Sewage Microbial Community to Dilution-Induced Reductions in Diversity

The relationship between functional redundancy and microbial community structure–diversity was examined using laboratory incubations to ensure constant environmental conditions. Serial dilutions of a sewage microbial community were prepared, used to inoculate sterile sewage, and maintained in batch culture. Probability suggests that dilution of the initial community should remove rare organism types, creating mixtures of cells differing in diversity. Regrowth of the diluted mixtures generated communities similar in abundance but differing in community structure and relative diversity (as determined using two DNA fingerprinting techniques and dilution-to-extinction analysis of community-level physiological profiles). The in situ function of each regrown community was examined by monitoring the short-term uptake of five different ¹⁴C-labeled compounds (glucose, acetate, citrate, palmitic acid, and an amino acid mixture). No significant differences were detected between treatments in either the rate of uptake of a substrate or the efficiency with which each community assimilated each compound. The fact that the activity of the original community was the same as that of a community regrown from an inoculum containing fewer that 100 cells (10⁻⁶ dilution) indicates that functional redundancy was quite high in this system. For each organism type eliminated during the dilution process, at least one of the remaining types was able to provide the same function at the same level as the lost one. Further research is necessary to determine what impact this functional redundancy may have on overall ecosystem function and stability.     

Role of some fermentation parameters on cyclosporin A production by a new isolate of Aspergillus terreus

A local isolate of Aspergillus terreus was selected among different microorganisms as a new cyclosporin A (Cy A) producing culture. The formation of Cy A was investigated under different fermentation conditions (including selection of the cultivation medium, fermentation time course, inoculum nature, medium volume, agitation rate, pH value). Relatively high Cy A productivities were maintained when the fermentation process was carried out using a medium composed of (g/L): glucose, 50; bactopeptone, 10; KH(2)PO(4), 5; KCl, 2.5; pH 5.3, inoculated with 2% standard inoculum of 48 h age, shaken at 200 rpm for 10 days.