Kinetics of microbial cell growth

As a rate equation of microbial cell growth, the Monod equation is widely used. However, this equation cannot fully correspond to real courses of microbial cell growth in many batch cultivations. Especially, predicted values based on this equation do not agree with observed values in many continuous cultivations. In this paper, which introduces new concepts of critical concentration and coefficient of consumption activity, the growth rate equation which corresponds to the whole period including lag period is newly derived and characteristics of microbial cell growth in batch cultivation are clarified. Further, applying the new rate equation to continuous cultivation, a general equation with which to calculate cell concentration is derived and characteristics of microbial cell growth in continuous cultivation are clarified. The calculated values of cell concentration based on the new theory showed quite good agreement with the observed values in both batch and continuous cultivation.     

The influence of cultivation methods on Shewanella oneidensis physiology and proteome expression

High-throughput analyses that are central to microbial systems biology and ecophysiology research benefit from highly homogeneous and physiologically well-defined cell cultures. While attention has focused on the technical variation associated with high-throughput technologies, biological variation introduced as a function of cell cultivation methods has been largely overlooked. This study evaluated the impact of cultivation methods, controlled batch or continuous culture in bioreactors versus shake flasks, on the reproducibility of global proteome measurements in Shewanella oneidensis MR-1. Variability in dissolved oxygen concentration and consumption rate, metabolite profiles, and proteome was greater in shake flask than controlled batch or chemostat cultures. Proteins indicative of suboxic and anaerobic growth (e.g., fumarate reductase and decaheme c-type cytochromes) were more abundant in cells from shake flasks compared to bioreactor cultures, a finding consistent with data demonstrating that “aerobic” flask cultures were O₂ deficient due to poor mass transfer kinetics. The work described herein establishes the necessity of controlled cultivation for ensuring highly reproducible and homogenous microbial cultures. By decreasing cell to cell variability, higher quality samples will allow for the interpretive accuracy necessary for drawing conclusions relevant to microbial systems biology research.     

Degradation of mineral oils by a selected microbial association

A series of microbial associations capable of degrading various petroleum oils, emulsols, and crude oil were obtained by selection during periodic or continuous cultivation. The formation of these associations and oil-product degradation occurred most efficiently during aerobic flow cultivation. Under these conditions, oils were degraded by 92% on average. The microbial degradation of a petroleum oil depended on its brand, concentration, emulsification, and aeration.     

Effects of continuous cucumber cropping and alternative rotations under protected cultivation on soil microbial community diversity

The diversity of soil microbial communities as affected by continuous cucumber cropping and alternative rotations under protected cultivation were evaluated using community level physiological profiles (CLPP) and random amplified polymorphic DNA (RAPD) analysis. The soils were selected from six cucumber cropping systems, which cover two cropping practices (rotation and continuous cropping) and a wide spectrum for cucumber cropping history under protected cultivation. Shannon–Weaver index and multivariate analysis were performed to characterize variations in soil microbial communities. Both CLPP and RAPD techniques demonstrated that cropping systems and plastic-greenhouse cultivation could considerably affect soil microbial functional diversity and DNA sequence diversity. The open-field soil had the highest Shannon–Weaver index (3.27 for CLPP and 1.50 for RAPD), whereas the lowest value occurred in the 7-year continuous protected cultivation soil (3.27 for CLPP and 1.50 for RAPD). The results demonstrated that continuous plastic-greenhouse cultivation and management can cause the reduction in the species diversity of the biota. Higher Shannon–Weaver index and coefficients of DNA sequence similarity were found in soils under rotation than those under continuous cropping. Cluster analysis also indicated that microbial community profiles of continuous cultivation soils were different from profiles of rotation soils. The reduction in diversity of microbial communities found in continuous cultivation soils as compared with rotation soils might be due to the differences in the quantity, quality and distribution of soil organic matter. Section Editor: D. E. Crowley     

Degradation of Petroleum Oils by a Selected Microbial Association

A series of microbial associations capable of the biodegradation of various petroleum oils, emulsols, and crude oil were obtained by selection during periodic or continuous cultivation. Formation of the associations and oil-product degradation occurred most efficiently during aerobic flow cultivation. Under these conditions, oils were degraded by 92% on average. The microbial degradation of a petroleum oil depended on its brand, concentration, emulsification, and aeration.     

Cultivating the uncultured: limits, advances and future challenges

Since the invention of the Petri dish, there have been continuous efforts to improve efficiency in microbial cultivation. These efforts were devoted to the attainment for diverse growth conditions, simulation of in situ conditions and achievement of high-throughput rates. As a result, prokaryotes catalysing novel redox reactions as well as representatives of abundant, but not-yet cultured taxa, were isolated. Significant insights into microbial physiology have been made by studying the small number of prokaryotes already cultured. However, despite these numerous breakthroughs, microbial cultivation is still a low-throughput process. The main hindrance to cultivation is likely due to the prevailing lack of knowledge on targeted species. In this review, we focus on the limiting factors surrounding cultivation. We discuss several cultivation obstacles, including the loss of microbial cell–cell communication following species isolation. Future research directions, including the refinement of culture media, strategies based on cell–cell communication and high-throughput innovations, are reviewed. We further propose that a combination of these approaches is urgently required to promote cultivation of uncultured species, thereby dawning a new era in the field.     

消毒、伴生和微生物菌对连作基质性质及黄瓜生长发育的影响

以连续种植2茬的连作基质为对象,采用室内盆栽试验,设置连作基质太阳能消毒、热水消毒、多菌灵消毒、大蒜伴生栽培和施入微生物菌处理以及新基质对照,在种植黄瓜后观测基质理化性质、养分含量、酚类物质含量、相关酶活性的变化以及黄瓜结果期生长发育、光合作用和果实产量的变化,探究不同处理对连作基质栽培黄瓜生长发育影响的机制,以期在生产上为废旧基质重复利用提供新的思路和方法。结果表明:在连作基质栽培黄瓜后,太阳能消毒和热水消毒处理均能显著提高连作基质的pH,同时降低电导率;施入微生物菌处理的基质EC值、速效氮、速效磷、速效钾和总酚含量均显著低于连作基质,而其基质磷酸酶活性和过氧化氢酶活性显著高于连作基质。热水消毒还显著降低连作基质的总酚含量和复合态酚含量。黄瓜生长至结果期,太阳能消毒、热水消毒、多菌灵消毒、大蒜伴生栽培和施入微生物菌均显著增强黄瓜的根系活力和株高;其中热水消毒和施入微生物菌处理的产量增加最为显著,分别比连作基质栽培提高了16.56%和18.40%。可见,连作基质进行热水消毒处理和施入微生物菌处理均能降低基质盐分积累,减少总酚含量,进而显著提高黄瓜的根系活力和光合作用,从而增加产量。     

Growth of yeast Candida utilis in crotonaldehyde containing media

Summary The inhibitory influence of the higher concentration of 20butenal, crotonaldehyde was followed during the batch and long-term continuous fermentation of Candida utilis growing on synthetic ethanol. Most crotonaldehyde is removed from the medium by biotransformation. Crotonaldehyde inhibits the growth, lengthens the lag phase and decreases the biomass yield and the content of crude proteins in the biomass. The yeast C. utilis is capable of growing on media containing very high concentrations of inhibitor in the in-flow during continuous cultivation. Uncharacteristic transport oscillations of the content of crotonaldehyde were observed for which acidic groups on the cell membrane are probably responsible. A sensitive method which is suitable for measuring very low concentration of crotonaldehyde in aqueous solutions is described. Crotonaldehyde acts as an uncompetitive inhibitor with slight mixed type of inhibition. An equation describing the kinetics of inhibition was derived.     

Directed autoselection of bacteria during turbidostat cultivation]

A method has been proposed for directed bacteria autoselection, based on their sensitivity to growth inhibitors during continuous unlimited cultivation. A smooth increase of a growth inhibitor concentration in the medium has been used in response to the appearance and autoselection of more resistant strains. The aim can be reached by continuous measurements of the specific microbial growth rate during the process and its keeping at the required level by the inhibitor additions. The experimental data are given showing application of the method for Escherichia coli and Pseudomonas sp. turbidostat cultivation with valine, low pH and formaldehyde as inhibitors.     

Enhanced microbial lipid production by <Emphasis Type="Italic">Cryptococcus albidus</Emphasis> in the high-cell-density continuous cultivation with membrane cell recycling and two-stage nutrient limitation

As a potential feedstock for biofuel production, a high-cell-density continuous culture for the lipid production by Cryptococcus albidus was investigated in this study. The influences of dilution rates in the single-stage continuous cultures were explored first. To reach a high-cell-density culture, a single-stage continuous culture coupled with a membrane cell recycling system was carried out at a constant dilution rate of 0.36/h with varied bleeding ratios. The maximum lipid productivity of 0.69 g/L/h was achieved with the highest bleeding ratio of 0.4. To reach a better lipid yield and content, a two-stage continuous cultivation was performed by adjusting the C/N ratio in two different stages. Finally, a lipid yield of 0.32 g/g and lipid content of 56.4% were obtained. This two-stage continuous cultivation, which provided a higher lipid production performance, shows a great potential for an industrial-scale biotechnological production of microbial lipids and biofuel production.     

Preservation of immobilized bacterial cell-matrix by drying for direct use in microbial sensors

A simple and effective method for the drying of immobilized bacterial cells to be used directly in a microbial biosensor for measurement of activity is reported. As a case example, plasmid-bearing cells of Alcaligenes eutrophus JMP 134, DSM 4058 were immobilized on various carriers and liquid-dried. The dried cell-matrix was used directly after rehydration/reactivation as the biological component of a biosensor for determining the concentration of xenobiotic compounds in the environment. Good viability results were obtained after long-term storage and cells exhibited no loss of plasmids responsible for the 2,4-dichlorophenoxyacetic acid (2,4-D) degradation. The activity of the cells for 2,4-D was proved using a respiration electrode. No time-consuming, repeated cell cultivation and harvesting was required, as the cells preserved from a single batch served as a continuous source for activity measurements. Many other microbial cultures can be preserved by this method and the cells preserved in the form of immobilized dried cell-matrix can be used directly to perform enzymatic tests, complex biochemical conversions and for production in the reactors. The dried cell-matrix can serve as a stable interchangeable component for a multipurpose biosensor.     

Production of Bacillus thuringiensis spores in total cell retention culture and two-stage continuous culture using an internal ceramic filter system

The production of Bacillus thuringiensis spores was investigated in a bioreactor incorporating a ceramic membrane filter to improve spore concentration and volumetric productivity. Two cultivation methods were used in this study: a total cell retention culture (TCRC), and a two-stage continuous culture with partial cell bleeding. In the TCRC, fed by 50 g/L of glucose, a spore concentration of 1.6 x 10(10) CFU/mL was obtained with a spore percentage of greater than 95% and a maximum cell mass of 82.2 g/L. The volumetric productivity was four times higher than that obtained from batch cultivation. In the two-stage continuous culture with partial cell bleeding spore concentration was strongly dependent on the bleed ratio. The spore concentration of 1.8 x 10(9) CFU/mL and the spore percentage of 70% were obtained at the second stage when a bleed ratio of 0.33 and a dilution rate of 0.23 h(-1) were used. (c) 1993 John Wiley & Sons, Inc.     

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Conventional microbial cell cultivation techniques are typically labor intensive, low throughput, and poorlyparallelized, rendering them inefficient. The development of automated, modular microbial cell micro-cultivation systems, particularly those employing droplet microfluidics, have gained attention for their high-throughput, highly paralellized and efficient cultivation capabilities. Here, we report the development of a microbial microdroplet culture system (MMC), which is an integrated platform for automated, high-throughput cultivation and adaptive evolution of microorganisms. We demonstrated that the MMC yielded both accurate and reproducible results for the manipulation and detection of droplets. The superior performance of MMC for microbial cell cultivation was validated by comparing the growth curves of six microbial strains grown in MMC, conventional shake flasks or well plates. The highest incipient growth rate for all six microbial strains was achieved by using MMC. We also conducted an 18-day process of adaptive evolution of methanol-essential Escherichia coli strain in MMC and obtained two strains exhibiting higher growth rates compared with the parent strain. Our study demonstrates the power of MMC to provide an efficient and reliable approach for automated, high-throughput microbial cultivation and adaptive evolution.     

Optimization of conditions for cell cultivation of Porphyra haitanensis conchocelis in a bubble-column bioreactor

Summary Process conditions for cell cultures derived from conchocelis of female red macroalga Porphyra haitanensis were optimized in an illuminated 0.3-l bubble-column photobioreactor, using CO₂ in air as the sole carbon source during a 20-day cultivation period. It reached the highest growth rate when the initial cell density was 700 mg l⁻¹ (dry weight), the optional aeration rate was 1.2 v/v/min, inorganic nitrate concentration was 15 mM and inorganic phosphate concentration was 0.6 mM. This is the first reported bioreactor cultivation study of cell cultures derived from conchocelis of Porphyra haitanensis.     

Cultivation of Neisseria meningitidis serogroup A on semisynthetic liquid nutrient media]

In cultivation of meningococcus of serological group A in fluid semisynthetic medium of simple composition prepared on the basis of purified acid casein hydrolysate with profound splitting there were obtained microbial cultures with a density of 4-5 x 10(9) microbial cells per 1 ml after 20-24 hours of cultivation with shaking. Alkalinity of the medium increased (to pH 8.0-8.2 during the stationary phase) with increase of the microbial cell concentration. A study of the accumulation of group-specific thermostable polysaccharide antigen in dynamics of meningococcus cultivation on semisynthetic medium tested showed the preparations obtained by alcoholic precipitation to be colourless and to contain much antigen (by inhibition of indirect hemagglutination), particularly at the phasees of negative growth acceleration and at the stationary phase. The suggested fluid semisynthetic medium of simple composition could be used for production of diagnostic and prophylactic meningococcus preparations belonging to the serological group A.     

High production of acetone-butanol-ethanol with high cell density culture by cell-recycling and bleeding

A continuous acetone-butanol-ethanol (ABE) production system with high cell density obtained by cell-recycling of Clostridium saccharoperbutylacetonicum N1-4 has been studied. In conventional continuous culture of ABE without cell-recycling, the cell concentration was below 5.2 g l(-1) and the maximum ABE productivity was only 1.85 g l(-1)h(-1) at a dilution rate of 0.20 h(-1). To obtain a high cell density at a faster rate, we concentrated the solventogenic cells of the broth 10 times by membrane filtration and were able to obtain approximately 20 g l(-1) of active cells after only 12h of cultivation. Continuous culture with cell-recycling was then started, and the cell concentration increased gradually through cultivation to a value greater than 100 g l(-1). The maximum ABE productivity of 11.0 gl(-1)h(-1) was obtained at a dilution rate of 0.85 h(-1). However, a cell concentration greater than 100 gl(-1) resulted in heavy bubbling and broth outflow, which made it impossible to carry out continuous culture. Therefore, to maintain a stable cell concentration, cell-bleeding was performed together with cell-recycling. At dilution rates of 0.11h(-1) and above for cell-bleeding, continuous culture with cell-recycling could be operated for more than 200 h without strain degeneration and the overall volumetric ABE productivity of 7.55 gl(-1)h(-1) was achieved at an ABE concentration of 8.58 gl(-1).     

Microorganisms destroying a number of nonionogenic surface-active substances

Microorganisms destroying nonionogenic surfactants (NS) were selected. The isolated cultures were assigned to the genera Pseudomonas and Bacillus. Biodestruction of 19 NS was studied in Erlenmeyer flasks with shaking and in the course of continuous cultivation. The rate of destruction was shown to depend on the chemical structure of a substance: block-copolymers of ethylene and propylene oxides were more resistant to the action of microorganisms. The results indicate that sewage waters containing NS (at an initial concentration of 50 to 500 mg/l) can be purified on local installations using microbial cultures or their complexes.     

Characterization of phototrophic purple nonsulfur bacteria forming colored microbial mats in a swine wastewater ditch

The community structure of pink-colored microbial mats naturally occurring in a swine wastewater ditch was studied by culture-independent biomarker and molecular methods as well as by conventional cultivation methods. The wastewater in the ditch contained acetate and propionate as the major carbon nutrients. Thin-section electron microscopy revealed that the microbial mats were dominated by rod-shaped cells containing intracytoplasmic membranes of the lamellar type. Smaller numbers of oval cells with vesicular internal membranes were also found. Spectroscopic analyses of the cell extract from the biomats showed the presence of bacteriochlorophyll a and carotenoids of the spirilloxanthin series. Ubiquinone-10 was detected as the major quinone. A clone library of the photosynthetic gene, pufM, constructed from the bulk DNA of the biomats showed that all of the clones were derived from members of the genera Rhodobacter and Rhodopseudomonas. The dominant phototrophic bacteria from the microbial mats were isolated by cultivation methods and identified as being of the genera Rhodobacter and Rhodopseudomonas by studying 16S rRNA and pufM gene sequence information. Experiments of oxygen uptake with lower fatty acids revealed that the freshly collected microbial mats and the Rhodopseudomonas isolates had a wider spectrum of carbon utilization and a higher affinity for acetate than did the Rhodobacter isolates. These results demonstrate that the microbial mats were dominated by the purple nonsulfur bacteria of the genera Rhodobacter and Rhodopseudomonas, and the bioavailability of lower fatty acids in wastewater is a key factor allowing the formation of visible microbial mats with these phototrophs.     

Modeling 1,2-dichloroethane biodegradation by <Emphasis Type="Italic">Klebsiella oxytoca va 8391</Emphasis> immobilized on granulated activated carbon

A mathematical model of the biodegradation of xenobiotics by microbial cells attached to particles of granulated activated carbon was developed. The model allowed the quantitative evaluation of different characteristics of the biofilm behavior: retarded microbial growth, increased concentration of immobilized cells compared to suspended cultures, potential cell detachment from the solid support and consequent independent growth of free cells. The applicability of the model was demonstrated for our own experimental data for 1,2- dichloroethane (DCA) biodegradation by Klebsiella oxytoca VA 8391 cells attached to granulated activated carbon. Two types of reactors, recirculated batch and continuous flow bioreactor, were studied. It was shown that in all investigated cases, the major contribution to DCA biodegradation was provided by the immobilized cells. Furthermore, immobilized cells were found to tolerate much higher substrate concentration and dilution rates in continuous culture than the free cells.     

设施土壤生态环境特点与调控

论述了设施土壤生态环境的特性主要是土壤养分富集 ,土壤板结 ;土壤酸化、次生盐渍化和连作障碍严重 ;土壤产生有害气体 ;土壤微生物区系变化很大。这些特点主要是盲目大量施肥 ,土壤长期连续利用 ,土壤环境密闭 ,单一化栽培 ,用肥种类不合理及某些化学物质积累造成的。利用生物、农业耕作、地上环境和工程措施调控相结合 ,是调控设施土壤生态环境的有效方法 ,土壤生物学的应用将是改善设施土壤生态环境新的研究方向。