Heterogeneity of Aspergillus niger microcolonies in liquid shaken cultures

The fungus Aspergillus niger forms (sub)millimeter microcolonies within a liquid shaken culture. Here, we show that such microcolonies are heterogeneous with respect to size and gene expression. Microcolonies of strains expressing green fluorescent protein (GFP) from the promoter of the glucoamlyase gene glaA or the ferulic acid esterase gene faeA were sorted on the basis of diameter and fluorescence using the Complex Object Parametric Analyzer and Sorter (COPAS) technology. Statistical analysis revealed that the liquid shaken culture consisted of two populations of microcolonies that differ by 90 μm in diameter. The population of small microcolonies of strains expressing GFP from the glaA or faeA promoter comprised 39% and 25% of the culture, respectively. Two populations of microcolonies could also be distinguished when the expression of GFP in these strains was analyzed. The population expressing a low level of GFP consisted of 68% and 44% of the culture, respectively. We also show that mRNA accumulation is heterogeneous within microcolonies of A. niger. Central and peripheral parts of the mycelium were isolated with laser microdissection and pressure catapulting (LMPC), and RNA from these samples was used for quantitative PCR analysis. This analysis showed that the RNA content per hypha was about 45 times higher at the periphery than in the center of the microcolony. Our data imply that the protein production of A. niger can be improved in industrial fermentations by reducing the heterogeneity within the culture.     

Correlation of actinomycin X2 to the lipid profile in static and shaken cultures of Streptomyces nasri strain YG62

Streptomyces nasri strain YG62 produces a broad-spectrum antibiotic designated actinomycin X2. The influence of static and shaken incubation on the production of actinomycin X2 and lipid profiles of S. nasri strain YG62 was investigated. It was found that shaken incubation was superior to the static process for both actinomycin X2 (2-fold) and total lipids (1.6-fold). Triglyceride and phospholipid levels paralleled the actinomycin X2 production with an increase in the triglyceride (2.8-fold) and phospholipid (1.2-fold) concentrations in the shaken culture over the static incubation. Analysis of fatty acid patterns revealed the occurrence of a wide range of fatty acids (C10-C22). The mean percentage of total saturated fatty acids in shaken culture was higher than those of the static culture. The mean percentage of mono-unsaturated fatty acids was almost the same in both cultures. The mean percentage of the total polyunsaturated fatty acids in the static culture was slightly higher than that of the shaken culture. The polyunsaturated/saturated fatty acid ratio (P/S) was higher in the static culture compared with the shaken culture. A positive correlation was recorded between triglycerides, phospholipids and actinomycin X2. A negative correlation on the other hand, was found between fatty acids and actinomycin X2.     

Fat synthesis from supplemented beet molasses by penicillium soppi zaleski in still and shaken cultures

Media composed of molasses alone did not support good growth nor high fat formation even when used at high concentrations. Peptone supplement as an external source of nitrogen accelerated both growth and fat formation in still as well as in shake cultures. But shaking mankedly suppressed growth and fat formation in media of molasses alone or molasses and peptone.Addition of corn steep liquor (CSL) to molasses gave rise in still cultures to rapid growth that was further accelerated by shaking. Effect of CSL supplement on fat formation, as measured by fat percentage in dry mycelium, was suppressive in still cultures. In shaken cultures, fat formation was promoted by the lowest concentration only. With higher concentrations of CSL, fat content increased but fat percentage in mycelium decreased.     

Introduction to the problems of differentiation in microorganisms

Folia Microbiologica -     

Isolation and structure elucidation of aza-sesquiterpenoids of protoilludane origin formed by shaken cultures of the fungus Clavicorona divaricata

Two novel sesquiterpenes of protoilludane origin, the alkaloids divaricatine C and D, have been isolated from MPGB shaken cultures of the fungus Clavicorona divaricata (Basidiomycetes). Their structures were elucidated by means of NMR studies and chemical correlations. The metabolites were weakly active against bacteria and inhibited the germination of the water cress Lepidium sativum. A possible mechanism of their formation from the protoilludane tsugicoline A (2) is suggested.     

A novel fed-batch based cultivation method provides high cell-density and improves yield of soluble recombinant proteins in shaken cultures

Background  

Cultivations for recombinant protein production in shake flasks should provide high cell densities, high protein productivity per cell and good protein quality. The methods described in laboratory handbooks often fail to reach these goals due to oxygen depletion, lack of pH control and the necessity to use low induction cell densities. In this article we describe the impact of a novel enzymatically controlled fed-batch cultivation technology on recombinant protein production in Escherichia coli in simple shaken cultures.     

Culture conditions promoting dispersed growth and biphasic production of actinorhodin in shaken cultures of Streptomyces coelicolor A3(2)

Media and culture conditions were developed for experiments on the physiology of secondary metabolism in Streptomyces coelicolor A3(2). Well dispersed mycelial growth was obtained in a buffered starch-glutamate-salts medium; a high (5%) starch concentration and addition of glass beads aided dispersal. Under the conditions developed, production of actinorhodin was suppressed during trophophase growth and began abruptly near the growth maximum.     

Genes, ageing and longevity in humans: problems, advantages and perspectives

Many epidemiological data indicate the presence of a strong familial component of longevity that is largely determined by genetics, and a number of possible associations between longevity and allelic variants of genes have been described. A breakthrough strategy to get insight into the genetics of longevity is the study of centenarians, the best example of successful ageing. We review the main results regarding nuclear genes as well as the mitochondrial genome, focusing on the investigations performed on Italian centenarians, compared to those from other countries. These studies produced interesting results on many putative “longevity genes”. Nevertheless, many discrepancies are reported, likely due to the population-specific interactions between gene pools and environment. New approaches, including large-scale studies using high-throughput techniques, are urgently needed to overcome the limits of traditional association studies performed on a limited number of polymorphisms in order to make substantial progress to disentangle the genetics of a trait as complex as human longevity.     

Use of fractal theory in neuroscience: methods, advantages, and potential problems

Fractal analysis has already found widespread application in the field of neuroscience and is being used in many other areas. Applications are many and include ion channel kinetics of biological membranes and classification of neurons according to their branching characteristics. In this article we review some practical methods that are now available to allow the determination of the complexity and scaling relationships of anatomical and physiological patterns. The problems of describing fractal dimensions are discussed and the concept of fractal dimensionality is introduced. Several related methodological considerations, such as preparation of the image and estimation of the fractal dimensions from the data points, as well as the advantages and problems of fractal geometric analysis, are discussed.     

Planar arrangement of eukaryotic cells in merged hydrogels combines the advantages of 3-D and 2-D cultures

We report an unordered 2-D array of eukaryotic cells completely embedded in a 3-D matrix. Every cell is located at the same distance from the gel surface, which ensures uniformity of growth conditions and ease of observation characteristic of a 2-D culture. Each cell is firmly immobilized, and each has a unique address in the array. The cells can be rapidly screened, individually monitored during extended time periods, and cultured with the formation of spheroid microcolonies characteristic of a 3-D culture. Individual microcolonies can be extracted from the gel and further propagated, thus enabling isolation of pure cell clones from rather dense cell populations and rapid drug-free generation of stable cell lines.     

Immobilization of lipases on hydrophobic supports: immobilization mechanism,advantages, problems,and solutions

Lipases are the most widely used enzymes in biocatalysis, and the most utilized method for enzyme immobilization is using hydrophobic supports at low ionic strength. This method allows the one step immobilization, purification, stabilization, and hyperactivation of lipases, and that is the main cause of their popularity. This review focuses on these lipase immobilization supports. First, the advantages of these supports for lipase immobilization will be presented and the likeliest immobilization mechanism (interfacial activation on the support surface) will be revised. Then, its main shortcoming will be discussed: enzyme desorption under certain conditions (such as high temperature, presence of cosolvents or detergent molecules). Methods to overcome this problem include physical or chemical crosslinking of the immobilized enzyme molecules or using heterofunctional supports. Thus, supports containing hydrophobic acyl chain plus epoxy, glutaraldehyde, ionic, vinylsulfone or glyoxyl groups have been designed. This prevents enzyme desorption and improved enzyme stability, but it may have some limitations, that will be discussed and some additional solutions will be proposed (e.g., chemical amination of the enzyme to have a full covalent enzyme-support reaction). These immobilized lipases may be subject to unfolding and refolding strategies to reactivate inactivated enzymes. Finally, these biocatalysts have been used in new strategies for enzyme coimmobilization, where the most stable enzyme could be reutilized after desorption of the least stable one after its inactivation.     

Biochemical and physical changes in shaken suspensions of Pasteurella pestis

Pasteurella pestis, harvested after 24 to 30 hr of growth in a casein hydrolysate medium at 26 C, was resuspended and shaken in 3% lactose-0.1 m phosphate buffer for 4 hr at the same temperature. Certain characteristics of these starved cells were compared with those of control cells. No differences in the amounts of cellular carbohydrate or lipid were detected. The concentrations of the principal free amino acids were greater in the shaken cells, except that they contained no measureable arginine, and the normally large pools of intracellular tricarboxylic acid cycle intermediates were reduced. Greater viable-cell counts resulted with the cells that were shaken in lactose buffer than with the control cells when each was incubated at 5 C for several weeks. However, the reduced viabilities were apparent losses caused by the formation of aggregates of cells. The clumping of cells was caused by the polymerization of extracellular nucleic acids, principally deoxyribonucleic acid, that were excreted by the cells. Cell clumping could be partially prevented by prior shaking of the suspended cells, which removed some of the deleterious material, or by the action of crystalline deoxyribonuclease.