Improving the batch-to-batch reproducibility of microbial cultures during recombinant protein production by regulation of the total carbon dioxide production

Batch-to-batch reproducibility of fermentation processes performed during the manufacturing processes of biologics can be increased by operating the cultures at feed rate profiles that are robust against typically arising disturbances. Remaining randomly appearing deviations from the desired path should be suppressed automatically by manipulating the feed rate. With respect to the cells' physiology it is best guiding the cultivations along an optimal profile of the specific biomass growth rate mu(t). However, there are two problems that speak for further investigations: Upon severe disturbances that may happen during the fermentation, the biomass concentration X may significantly deviate from its desired value, then a fixed mu-profile leads to a diminished batch-to-batch reproducibility. Second, the specific growth rate cannot easily be estimated online to a favourably high accuracy, hence it is difficult to determine the deviations in mu from the desired profile. The alternative discussed here solves both problems by keeping the process at the corresponding total cumulative carbon dioxide production-profile: it is robust against distortions in X and the controlled variable can accurately be measured online during cultivations of all relevant sizes. As compared to the fermentation practice currently used in industry, the experimental results, presented at the example of a recombinant protein production with Escherichia coli cells, show that CPR-based corrections lead to a considerably improved batch-to-batch reproducibility.     

Harvest mice<Emphasis Type="Italic">Micromys minutus</Emphasis> and common dormice<Emphasis Type="Italic">Muscardinus avellanarius</Emphasis> live sympatric in woodland habitat

In an overgrown clearing, which occupied an area of 5 ha within mixed spruce-deciduous forest, 106 and 20 nests of the harvest mouseMicromys minutus (Pallas, 1771) and 81 and 59 nests of the common dormouseMuscardinus avellanarius (Linnaeus, 1758) were recorded in the fourth and fifth years after clear-felling, respectively. The highest densities of nests ofM. minutus andM. avellanarius were 46 nests/ha and 39 nests/ha, respectively, in two different plots. The affinity betweenM. minutus andM. avellanarius was negative in overgrown clearings according to the distribution of their nests. Such a result was expected becauseM. minutus andM. avellanarius used different nest supporting plants:M. minutus used tall grasses, whileM. avellanarius used young trees and shrubs. However, no positive relationship was found between the number of nests ofM. minutus and cover of grass vegetation in plots with the highest density of nests ofM. minutus. Most nests ofM. Minutus were situated in areas covered by young trees among which tall grasses, mainlyCalamagrostis epigeios, grew, often on the borders with the areas covered by grass vegetation. The successionary stage when woody vegetation reached 4–5 years old did not choke grass vegetation yet was favourable for bothM. minutus andM. avellanarius in overgrowing clearings.     

The influence of high nestbox density on the common dormouseMuscardinus avellanarius population

The response of common dormouseMuscardinus avellanarius Linnaeus, 1758 population to availability of nest sites was studied by manipulating the nestbox grid and ring-marking dormice. Abundance of adult dormice more than doubled in the 25 × 25 m nestbox grid in comparison to the 50 × 50 m grid, as a result of increased nestbox density from four to 16 boxes/ha. This effect already became apparent in the first year after additional nestboxes were made available and resulted from dormouse immigration, mostly from adjacent areas without nestboxes. In the second and third years, the number of two-year-old and older resident dormice, which had their home ranges in this plot, increased considerably. The average size of dormouse home range decreased by approximately half both in males and females in the 25 × 25 m grid compared to the 50 × 50 m grid. The proportion of breeding adult females did not differ between the two grids in spite of different adult dormouse density. Shortage of secure nest sites was a limiting factor for the common dormouse population abundance in the forest where natural tree hollows were absent, and high nestbox density increased environmental carrying capacity.     

Control of cultivation processes for recombinant protein production: a review

The current state-of-the-art in control of cultivation processes for recombinant protein production is examined including the quantitative knowledge that can be activated for this purpose and the measurement techniques that can be employed for control at industrial manufacturing sites.     

Data-based optimization of protein production processes

While data-based modeling is possible in various ways, data-based optimization has not been previously described. Here we present such an optimization technique. It is based on dynamic programming principles and uses data directly from exploratory experiments where the influence of the adjustable variables u were tested at various values. Instead of formulating the performance index J as a function of time t within a cultivation process it is formulated as a function of the biomass x. The advantage of this representation is that in most biochemical production processes J(x) only depends of the vector u of the adjustable variables. This given, mathematical programming techniques allow determining the desired optimal paths u _opt (x) from the x-derivatives of J(x). The resulting u _opt (x) can easily be transformed back to the u(t) profiles that can then be used in an improved fermentation run. The optimization technique can easily be explained graphically. With numerical experiments the feasibility of the method is demonstrated. Then, two optimization runs for recombinant protein formations in E. coli are discussed and experimental validation results are presented.     

Clonality in the postfire root rot ascomycete Rhizina undulata

The aim of the present work was to investigate the potential for territorial and dispersive clonality in natural populations of the postfire root rot ascomycete Rhizina undulata. Population studies based on vegetative compatibility tests were done with strains isolated from individual sporocarps at five burned sites in three different localities (separated by 20-40 km) in the Curronian Spit of western Lithuania. Among a total of 103 strains, the tests identified 14 distinct vegetative compatibility groups (VCGs) of R. undulata, 13 of which were represented by 2-48 strains and three were encountered at 2-4 different sites. Occurrence on spatially separated sites of the same VCG of the fungus indicated a presence of dispersive clonality in R. undulata populations. On a local scale clusters of vegetative compatible sporocarps usually occupied discrete territories, implying territorial clonality. The two largest VCGs covered areas up to 7 and 3 m across. The results show that both dispersive and territorial clones are characteristics of natural populations of the fungus.     

Frequency and demographic parameters of white-tipped common dormiceMuscardinus avellanarius

Out of 1154 marked common dormiceMuscardinus avellanarius (Linnaeus, 1758), 219 animals (19.0%) had white tail tips at Vilkaraistis forest (Moletai district, east Lithuania) in 1984–1993. The extent of white tail-tip varied from a slight tuft to a notable 22 mm length of white hair in the tail tip. In this population white-tipped individuals represented, on average, 25.7% among juvenile females, 24.9% among adult females, 13.4% among juvenile males, and 15.7% among adult males. White-tipped females were significantly more frequent than males, both in juveniles and in adults. The share of white-tipped dormice fluctuated in different years from 12.5% to 25.6% among adult dormice, and from 9.2% to 28.3% among juveniles. The percentage of white-tipped dormice among juveniles in different years was directly proportional to population density in autumn. There was no clear difference or trend in the mortality rate in white-tipped versus normal dormice. The contribution of white-tipped females to population reproduction was relatively lower than that of normal females: only 18.4% out of all registered breeding cases (n=212) were due to white-tipped females.     

Phylogenetic relationships in European Ceriporiopsis species inferred from nuclear and mitochondrial ribosomal DNA sequences

The aim of this work was to clarify taxonomy and examine evolutionary relationships within European Ceriporiopsis species using a combined analysis of the large subunit (nLSU) nuclear rRNA and small subunit (mtSSU) mitochondrial rRNA gene sequences. Data from the ITS region were applied to enhance the view of the phylogenetic relationships among different species. The studied samples grouped into four complex clades, suggesting that the genus Ceriporiopsis is polyphyletic. The generic type Ceriporiopsis gilvescens formed a separate group together with Ceriporiopsis guidella and Phlebia spp. in the phlebioid clade. In this clade, the closely related species Ceriporiopsis resinascens and Ceriporiopsis pseudogilvescens grouped together with Ceriporiopsis aneirina. C. resinascens and C. pseudogilvescens have identical LSU and SSU sequences but differ in ITS. Ceriporiopsis pannocincta also fell in the phlebioid clade, but showed closer proximity to Gloeoporus dichrous than to C. gilvescens or C. aneirina–C. pseudogilvescens–C. resinascens group. Another clade was composed of a Ceriporiopsis balaenae–Ceriporiopsis consobrina group and was found to be closely related to Antrodiella and Frantisekia, with the overall clade highly reminiscent of the residual polyporoid clade. The monotypic genus Pouzaroporia, erected in the past for Ceriporiopsis subrufa due to its remarkable morphological differences, also fell within the residual polyporoid clade. Ceriporiopsis subvermispora held an isolated position from the other species of the genus. Therefore, the previously proposed name Gelatoporia subvermispora has been adopted for this species. Physisporinus rivulosus appeared unrelated to two other European Physisporinus species. Moreover, Ceriporiopsis (=Skeletocutis) jelicii grouped in a separate clade, distinct from Ceriporiopsis species. Finally, the ITS data demonstrated the proximity of some Ceriporiopsis species (Ceriporiopsis portcrosensis and Ceriporiopsis subsphaerospora) to Skeletocutis amorpha.     

High survival during hibernation affects onset and timing of reproduction

The timing of reproduction is one of the most crucial life history traits, with enormous consequences for the fitness of an individual. We investigated the effects of season and timing of birth on local survival probability in a small mammalian hibernator, the common dormouse (Muscardinus avellanarius). Local monthly survival probability was lowest in the early active season (May–August, ϕ_adult = 0.75–0.88, ϕ_juvenile = 0.61–0.68), increased during the late active season (August–October), and highest during hibernation (October–May, ϕ_adult = 0.96–0.98, ϕ_juvenile = 0.81–0.94). Consequently, dormice had an extremely high winter survival probability. We observed two peaks in the timing of reproduction (June and August/September, respectively), with the majority of juveniles born late in the active season. Although early investment in reproduction seems the better life history tactic [survival probability until onset of reproduction: ϕ_{born early} = 0.46, 95% confidence interval (CI) 0.28–0.64; ϕ_{born late} = 0.19, 95% CI = 0.09–0.28], only females with a good body condition (significantly higher body mass) invest in reproduction early in the year. We suggest the high over-winter survival in dormice allows for a unique life history pattern (i.e., combining slow and fast life history tactics), which leads to a bimodal seasonal birth pattern: (1) give birth as early as possible to allow even the young to breed before hibernating, and/or (2) give birth as late as possible (leaving just enough time for these young to fatten) and enter directly into a period associated with the highest survival rates (hibernation) until maturity.