Penicillin fermentation: mechanisms and models for industrial-scale bioreactors

Even after many years of research and industrial practice, the production of penicillin G in fed-batch fermentation by Penicillium crysogenum continues to attract research interest. There are many reasons: the commercial and therapeutic importance of penicillin and its derivatives, the complexity of cell growth, and the impact of engineering variables, the last of which are significant in large bioreactors but are not yet fully understood. Extensive research has generated new information on the mechanisms of cellular reactions and morphological features of the mycelia and their role in the synthesis of the product. Given a choice of mechanisms, models of different degrees of complexity, for both cellular differentiation and bioreactor performance, have been proposed. The more complex models require and provide more information. They are also more difficult to evaluate and apply in automatic control systems for production-scale bioreactors. The present review considers the evolution of recent knowledge and models from this perspective.     

Monitoring bioreactors using principal component analysis: production of penicillin G acylase as a case study

The complexity of biological processes often makes impractical the development of detailed, structured phenomenological models of the cultivation of microorganisms in bioreactors. In this context, data pre-treatment techniques are useful for bioprocess control and fault detection. Among them, principal component analysis (PCA) plays an important role. This work presents a case study of the application of this technique during real experiments, where the enzyme penicillin G acylase (PGA) was produced by Bacillus megaterium ATCC 14945. PGA hydrolyzes penicillin G to yield 6-aminopenicilanic acid (6-APA) and phenyl acetic acid. 6-APA is used to produce semi-synthetic β-lactam antibiotics. A static PCA algorithm was implemented for on-line detection of deviations from the desired process behavior. The experiments were carried out in a 2-L bioreactor. Hotteling’s T ² was the discrimination criterion employed in this multivariable problem and the method showed a high sensibility for fault detection in all real cases that were studied.     

Performance of industrial scale bioreactors with modified RUSHTON turbine agitators

The data presented here with respect to the behaviour of industrial scale stirred tank bioreactors equipped with modified RUSHTON turbine agitators in the biosynthesis processes of antibiotics are valid for that case that the power consumption is the same as it is in standard RUSHTON turbine agitators. Each modified RUSHTON turbine agitator was obtained through the variation of the blade surface by adding perforations so that the ratio between the perforation surface area and the full surface area (or the surface fraction of the perforations) is 0.36. In the fermentations of Streptomyces aureofaciens, Streptomyces rimosus and Penicillium chrysogenum producing tetracycline, oxytetracyline and penicillin, respectively, in bioreactors equipped with modified RUSHTON turbine agitators, the relative antibiotic production is increased by more than 30% compared to standard bioreactors.     

Cytoskeletal involvement in neuronal learning: a review

This paper introduces the ideas of neural networks in the context of currently recognized cellular structures within neurons. Neural network models and paradigms require adaptation of synapses for learning to occur in the network. Some models of learning paradigms require information to move from axon to dendrite. This motivated us to examine the possibility of intracellular signaling to mediate such signals. The cytoskeleton forms a substrate for intracellular signaling via material transport and other putative mechanisms. Furthermore, many experimental results suggest a link between the cytoskeleton and cognitive processing. In this paper we review research on intracellular signaling in the context of neural network learning.Abbreviations MT microtubule - MTs microtubules - ART adaptive resonance theory - RCE restricted coulomb energy - MAP microtubule associated protein - NO nitric oxide Correspondence to: J. Dayhoff     

A cellular automata model for simulating fed-batch penicillin fermentation process

A cellular automata model to simulate penicillin fed-batch fermentation process (CAPFM) was established in this study, based on a morphologically structured dynamic penicillin production model, that is in turn based on the growth mechanism of penicillin producing microorganisms and the characteristics of penicillin fed-batch fermentation. CAPFM uses the three-dimensional cellular automata as a growth space, and a Moore-type neighborhood as the cellular neighborhood. The transition rules of CAPFM are designed based on mechanical and structural kinetic models of penicillin batch-fed fermentation processes. Every cell of CAPFM represents a single or specific number of penicillin producing microorganisms, and has various state. The simulation experimental results show that CAPFM replicates the evolutionary behavior of penicillin batch-fed fermentation processes described by the structured penicillin production kinetic model accordingly. __________ Translated from ACTA BIOPHYSICA, 2005, 21(2) [译自: 生物物理学报, 2005,21(2)]     

Over‐pressurized bioreactors: Application to microbial cell cultures

In industrial biotechnology, microbial cultures are exposed to different local pressures inside bioreactors. Depending on the microbial species and strains, the increased pressure may have detrimental or beneficial effects on cellular growth and product formation. In this review, the effects of increased air pressure on various microbial cultures growing in bioreactors under moderate total pressure conditions (maximum, 15 bar) will be discussed. Recent data illustrating the diversity of increased air pressure effects at different levels in microbial cells cultivation will be presented, with particular attention to the effects of oxygen and carbon dioxide partial pressures on cellular growth and product formation, and the concomitant effect of oxygen pressure on antioxidant cellular defense mechanisms. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:767–775, 2014     

Streamlined production of genetically modified T cells with activation,transduction and expansion in closed-system G-Rex bioreactors

BackgroundGas Permeable Rapid Expansion (G-Rex) bioreactors have been shown to efficiently expand immune cells intended for therapeutic use, but do not address the complexity of the viral transduction step required for many engineered T-cell products. Here we demonstrate a novel method for transduction of activated T cells with Vectofusin-1 reagent. Transduction is accomplished in suspension, in G-Rex bioreactors. The simplified transduction step is integrated into a streamlined process that uses a single bioreactor with limited operator intervention.MethodsPeripheral blood mononuclear cells (PBMCs) from healthy donors were thawed, washed and activated with soluble anti-CD3 and anti-CD28 antibodies either in cell culture bags or in G-Rex bioreactors. Cells were cultured in TexMACS GMP medium with interleukin (IL)-7 and IL-15 and transduced with RetroNectin in bags or Vectorfusin-1 in the G-Rex. Total viable cell number, fold expansion, viability, transduction efficiency, phenotype and function were compared between the two processes.ResultsThe simplified process uses a single vessel from activation through harvest and achieves 56% transduction with 29-fold expansion in 11 days. The cells generated in the simplified process do not differ from cells produced in the conventional bag-based process functionally or phenotypically.DiscussionThis study demonstrates that T cells can be transduced in suspension. Further, the conventional method of generating engineered T cells in bags for clinical use can be streamlined to a much simpler, less-expensive process without compromising the quality or function of the cell product.     

Sampling bias in the determination of first and last occurrences

Background: Past studies of first and last occurrence dates of phenological events have revealed close associations with climatic parameters. Consequently, it is widely acknowledged that recent shifts in the beginning, duration or ending of such events are a response to present climate change. In addition, in recent times, there have tended to be many more observers than in earlier times, especially in urban areas. Furthermore, the number of individuals (plants or animals) observed has often changed markedly. In many situations it is not possible to obtain the average first or last occurrence date of a group of individuals, and only the most extreme occurrence is recorded. This common observational difficulty leads to sampling bias that needs to be taken into account.

Aim: Our aim is to use statistical models to quantify the sampling bias and its dependence on sample size and the variability and correlation amongst the individuals under consideration.

Methods: n-dimensional multivariate normal distribution and two-way fixed-effects analysis of variance models were developed to examine the dependence of the sampling bias on the above factors. Our results are compared with real data.

Results: For first and last occurrence observations, which are the most common index in many phenological studies, we found that changes in observational practice and sample size can, in certain circumstances, easily produce changes in bias that can swamp (or indeed reverse) any climatic change effects.

Conclusions: Our new, realistic statistical models allow the sampling bias to be quantified and calculated in terms of the number of individuals under observation, their variability and the degree of correlation between individuals.     

Application of Human‐Induced Pluripotent Stem Cells (hiPSCs) to Study Synaptopathy of Neurodevelopmental Disorders

Synapses are the basic structural and functional units for information processing and storage in the brain. Their diverse properties and functions ultimately underlie the complexity of human behavior. Proper development and maintenance of synapses are essential for normal functioning of the nervous system. Disruption in synaptogenesis and the consequent alteration in synaptic function have been strongly implicated to cause neurodevelopmental disorders such as autism spectrum disorders (ASDs) and schizophrenia (SCZ). The introduction of human‐induced pluripotent stem cells (hiPSCs) provides a new path to elucidate disease mechanisms and potential therapies. In this review, we will discuss the advantages and limitations of using hiPSC‐derived neurons to study synaptic disorders. Many mutations in genes encoding for proteins that regulate synaptogenesis have been identified in patients with ASDs and SCZ. We use Methyl‐CpG binding protein 2 (MECP2), SH3 and multiple ankyrin repeat domains 3 (SHANK3) and Disrupted in schizophrenia 1 (DISC1) as examples to illustrate the promise of using hiPSCs as cellular models to elucidate the mechanisms underlying disease‐related synaptopathy.     

Using concept maps to characterise cellular respiration knowledge in undergraduate students

ABSTRACT

Meaningful learning occurs by relating new information to and revising prior knowledge, making it essential to understand student knowledge before helping them move toward a more scientific understanding. In this study, we characterise prior knowledge about cellular respiration in undergraduate students enrolled in introductory biology by analysing student-constructed concept maps (N = 182) and interviews (N = 9). Students were instructed to create concept maps from a bank of 20 concepts with the purpose of interconnecting the processes of cellular respiration, showing how pools of ATP are generated and used, and identifying where the events of cellular respiration occur. Student maps were analysed for content, quality and organisation of knowledge. Interviews were used to corroborate inferences made from concept maps. Students had a simplified understanding of cellular respiration and its processes as evident by cognitive structures with limited quantities of schemas that were vaguely connected and linearly organised. Furthermore, students had a better understanding of glycolysis than fermentation. Instructors can use these findings to help students build better knowledge of cellular respiration by focusing on incorporating relevant schemas, creating quality connections among schemas, and organising their knowledge of cellular respiration to reflect biological complexity.     

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.     

Hairy Root Culture for Mass-Production of High-Value Secondary Metabolites

ABSTRACT

Plant cell cultivations are being considered as an alternative to agricultural processes for producing valuable phytochemicals. Since many of these products (secondary metabolites) are obtained by direct extraction from plants grown in natural habitat, several factors can alter their yield. The use of plant cell cultures has overcome several inconveniences for the production of these secondary metabolites. Organized cultures, and especially root cultures, can make a significant contribution in the production of secondary metabolites. Most of the research efforts that use differentiated cultures instead of cell suspension cultures have focused on transformed (hairy) roots. Agrobacterium rhizogenes causes hairy root disease in plants. The neoplastic (cancerous) roots produced by A. rhizogenes infection are characterized by high growth rate, genetic stability and growth in hormone free media. These genetically transformed root cultures can produce levels of secondary metabolites comparable to that of intact plants. Hairy root cultures offer promise for high production and productivity of valuable secondary metabolites (used as pharmaceuticals, pigments and flavors) in many plants. The main constraint for commercial exploitation of hairy root cultivations is the development and scaling up of appropriate reactor vessels (bioreactors) that permit the growth of interconnected tissues normally unevenly distributed throughout the vessel. Emphasis has focused on designing appropriate bioreactors suitable to culture the delicate and sensitive plant hairy roots. Recent reactors used for mass production of hairy roots can roughly be divided as liquid-phase, gas-phase, or hybrid reactors. The present review highlights the nature, applications, perspectives and scale up of hairy root cultures for the production of valuable secondary metabolites.     

A novel micromanipulation technique for measuring the bursting strength of single mammalian cells

Summary Information about the bursting strength of animal cells is essential if the mechanisms of cell damage in bioreactors are to be understood, and if cell mechanical properties are ever to be related to cell structure and physiology. We have developed a novel cell compression technique that makes it possible to directly measure the bursting strength of single mammalian cells, and to infer information about cell mechanical properties. Offprint requests to: C. R. Thomas     

Utilization of side-chain precursors for penicillin biosynthesis in a high-producing strain of Penicillium chrysogenum

Utilization of the side-chain precursors phenoxyacetic acid (POA) and phenylacetic acid (PA) for penicillin biosynthesis by Penicillium chrysogenum was studied in shake flasks. Precursor uptake and penicillin production were followed by HPLC analysis of precursors and products in the medium and in the cells. P. chrysogenum used both POA and PA as precursors, producing phenoxymethylpenicillin (penicillin V) and benzylpenicillin (penicillin G), respectively. If both precursors were present simultaneously, the formation of penicillin V was blocked and only penicillin G was produced. When PA was added at different times to cells that were induced initially for POA utilization and were producing penicillin V, the POA utilization and penicillin V formation were blocked, whereas the cells started utilizing PA and produced penicillin G. The blocking of the POA turnover lasted for as long as PA was present in the medium. If POA was added to cultures induced initially for PA utilization and producing penicillin G, this continued irrespective of the presence of POA. Utilization of POA increased concomitant with depletion of PA from the medium. Analysis of cellular pools from a growing cell system with POA as precursor to which PA was added after 48 h showed that the cellular concentration of POA was kept high without production of penicillin V and at a concentration comparable to the concentration in the medium. The cellular concentration of POA was higher than the concentration of PA that was utilized for penicillin G production. Correspondence to: S. Havn Eriksen     

Neurodegenerative mutants in Drosophila: a means to identify genes and mechanisms involved in human diseases?

There are 50 ways to leave your lover (Simon 1987) but many more to kill your brain cells. Several neurodegenerative diseases in humans, like Alzheimer’s disease, have been intensely studied but the underlying cellular and molecular mechanisms are still unknown for most of them. For those syndromes where associated gene products have been identified their biochemistry and physiological as well as pathogenic function is often still under debate. This is in part due to the inherent limitations of genetic analyses in humans and other mammals and therefore experimentally accessible invertebrate in vivo models, such as Caenorhabditis elegans and Drosophila melanogaster, have recently been introduced to investigate neurodegenerative syndromes. Several laboratories have used transgenic approaches in Drosophila to study the human genes associated with neurodegenerative diseases. This has added substantially to our understanding of the mechanisms leading to neurodegenerative diseases in humans. The isolation and characterization of Drosophila mutants, which display a variety of neurodegenerative phenotypes, also provide valuable insights into genes, pathways, and mechanisms causing neurodegeneration. So far only about two dozen such mutants have been described but already their characterization reveals an involvement of various cellular functions in neurodegeneration, ranging from preventing oxidative stress to RNA editing. Some of the isolated genes can already be associated with human neurodegenerative diseases and hopefully the isolation and characterization of more of these mutants, together with an analysis of homologous genes in vertebrate models, will provide insights into the genetic and molecular basis of human neurodegenerative diseases.     

A review of the threats to adult survival for swallows (Family: Hirundinidae)

Capsule: For declining migratory birds, including many aerial insectivores, such as swallows, there is evidence that adult survival is a demographic process with strong effects on population trends.

Aims: The aim was to identify and quantify the effect of threats affecting adult survival and potentially driving population declines for five well-studied swallow species: Barn Swallow Hirundo rustica, Cliff Swallow Petrochelidon pyrrhonota, Tree Swallow Tachycineta bicolor, Sand Martin Riparia riparia, and Purple Martin Progne subis.

Methods: We reviewed the literature to identify the threats to adult survival, quantified the magnitude of the effect and identified whether threats had a direct or indirect effect on survival.

Results: We identified habitat change, weather, competition, incidental loss, contaminants, insect availability, disease, and predation as threats to adult survival in swallows, although for many of these threats there was limited information to quantify their impact. However, weather, particularly cold snaps and precipitation, had negative effects on survival for many populations of four species, either directly or indirectly through effects on insect availability. When there was a relationship, weather was associated with a 13–53% decrease in survival.

Conclusion: Based on the available research, weather conditions throughout the annual cycle is a key threat to adult survival for several swallow species. However, future research on the threats to these species should consider examining the effect of insect availability and the effect of threats during the non-breeding period on survival. Finally, we suggest that new research should be devoted to understanding the importance of adult survival for declining bird populations.     

Progress in the understanding and treatment of Fabry disease

BackgroundFabry disease is caused by α-galactosidase A deficiency. Substrates of this lysosomal enzyme accumulate, resulting in cellular dysfunction. Patients experience neuropathic pain, kidney failure, heart disease, and strokes.Scope of reviewThe clinical picture and molecular features of Fabry disease are described, along with updates on disease mechanisms, animal models, and therapies.Major conclusionsHow the accumulation of α-galactosidase A substrates, mainly glycosphingolipids, leads to organ damage is incompletely understood. Enzyme replacement and chaperone therapies are clinically available to patients, while substrate reduction, mRNA-based, and gene therapies are on the horizon. Animal models exist to optimize these therapies and elucidate disease mechanisms for novel treatments.General significanceRecent newborn screening studies demonstrate that Fabry disease is the most common lysosomal storage disease. As many countries now include Fabry disease in their screening panels, the number of identified patients is expected to increase significantly. Better knowledge of disease pathogenesis is needed to improve treatment options.