Microbial cultivation in rocking single-use bioreactors

The application of single-use bioreactors (SUB) for microbial cultivation, especially of reactor designs beyond the traditional stirred tank, is usually regarded as crucial. Especially the usually low gas mass transfer coefficients are insufficient, however this is not true for 2-dimensionally rocking motion bioreactors like the CELL-tainer®. Volumetric gas mass transfer coefficients (kLa-values) of 600 h-1 are achieved (fig. 1), which allow bacterial fed-batch cultivations up to a cell density of 50 gL-1 at growth rates of 0.3 h-1 w/o any oxygen blending (Junne et al, Chem Eng Technol 2013, 85, p. 57-66). One major asset in this respect are the low maximum shear forces in comparison to stirred tank reactors. This feature might be beneficial when shear sensitive microbes are cultivated, like marine phototrophic and heterotrophic microalgae and filamentous organisms. Please click Additional Files below to see the full abstract

Designing a microbial cultivation platform for continuous biopharmaceutical production

The existing biopharmaceutical manufacturing paradigm is poorly suited to produce biologic drugs on demand at a point-of-care. Generally, commercial-scale (~2,000 - 10,000 L) manufacturing using fed-batch cultivation and fixed stainless-steel infrastructure is concentrated in developed nations and results in process cycle times on the order of weeks to months.1,2 Coupled with the complex logistical challenges associated with continuous “plant-to-patient” cold-chains, the geographically biased nature of therapeutic protein production today can limit access to biologic drugs in developing areas of the world.3 There is an opportunity to create technologies capable of rapidly generating biopharmaceuticals in situ in emergency situations, in remote healthcare settings, and in the battlefield. A platform that incorporates a modular suite of bioreactor, purification, and in-line analytics technologies has the potential to bridge this gap if developed in parallel with appropriately engineered stains of a flexible expression host. This poster will describe a multifaceted approach towards the development of a fully automated bench-scale perfusion process for the cultivation of Pichia pastoris and expression of therapeutically relevant heterologous proteins. We demonstrate the application of computational fluid dynamics (CFD) simulations to the optimization of the cultivation environment within our bench-top bioreactors. We further show that Pichia pastoris is amenable to secreting a variety of recombinant proteins spanning a range of preexisting drug classes (e.g. hormones, cytokines, monoclonal antibodies, vaccine antigens). Among these therapeutic proteins are molecules that require proper co-/post-translational processing for bioactivity. We envision that the development of P. pastoris strains with the capability to perform these critical processing steps in vivo will mitigate the need to chemically modify proteins post-expression and reduce the number of unit operations required in a typical upstream process

Adaptive set-point control system for microbial cultivation processes

A control system for set-point control of microbial cultivation process parameters is developed, in which a tendency model is applied for controller adaptation to process nonlinearity and time-varying operating conditions. The tendency model is updated on-line and introduced into control algorithm for prediction of steady-state control action and returning of feedback controller. The control system was tested for controlling dissolved oxygen concentration in batch operating mode bioreactor under extreme operating conditions. In simulation experiments, the control system demonstrates fast adaptation, robust behaviour and significant improvement in control performance compared to that of fixed gain controller

Reclamation of Marine Chitinous Materials for Chitosanase Production via Microbial Conversion by Paenibacillus macerans

[[abstract]]: Chitinous materials from marine byproducts elicit great interest among biotechnologists for their potential biomedical or agricultural applications. In this study, four kinds of marine chitinous materials (squid pens, shrimp heads, demineralized shrimp shells, and demineralized crab shells) were used to screen the best source for producing chitosanase by Paenibacillus macerans TKU029. Among them, the chitosanase activity was found to be highest in the culture using the medium containing squid pens as the sole carbon/nitrogen (C/N) source. A chitosanase which showed molecular weights at 63 kDa was isolated from P. macerans cultured on a squid pens medium. The purified TKU029 chitosanase exhibited optimum activity at 60 ◦C and pH 7, and was stable at temperatures under 50 ◦C and pH 3-8. An analysis by MALDI-TOF MS revealed that the chitosan oligosaccharides (COS) obtained from the hydrolysis of water-soluble chitosan by TKU029 crude enzyme showed various degrees of polymerization (DP), varying from 3–6. The obtained COS enhanced the growth of four lactic acid bacteria strains but exhibited no effect on the growth of E. coli. By specialized growth enhancing effects, the COS produced from hydrolyzing water soluble chitosan with TKU029 chitinolytic enzymes could have potential for use in medicine or nutraceuticals.[[sponsorship]]MOST[[notice]]補正完

A Multi-Platform Flow Device for Microbial (Co-) Cultivation and Microscopic Analysis

Novel microbial cultivation platforms are of increasing interest to researchers in academia and industry. The development of materials with specialized chemical and geometric properties has opened up new possibilities in the study of previously unculturable microorganisms and has facilitated the design of elegant, high-throughput experimental set-ups. Within the context of the international Genetically Engineered Machine (iGEM) competition, we set out to design, manufacture, and implement a flow device that can accommodate multiple growth platforms, that is, a silicon nitride based microsieve and a porous aluminium oxide based microdish. It provides control over (co-)culturing conditions similar to a chemostat, while allowing organisms to be observed microscopically. The device was designed to be affordable, reusable, and above all, versatile. To test its functionality and general utility, we performed multiple experiments with Escherichia coli cells harboring synthetic gene circuits and were able to quantitatively study emerging expression dynamics in real-time via fluorescence microscopy. Furthermore, we demonstrated that the device provides a unique environment for the cultivation of nematodes, suggesting that the device could also prove useful in microscopy studies of multicellular microorganisms

Production of Microbial Lipids from Lignocellulosic Biomass

The current industrial production of the biodiesel relies mainly on vegetable oils that could result in the shortage of edible oils in food markets and increase in their prices. Microbial lipids produced by oleaginous microorganism have attracted a lot of attention in the recent years as a source of high-value polyunsaturated acids as well as alternative feedstock for the production of biodiesel. However, the production of microbial oils faces a number of problems concerning the costs of lipid extraction, carbon source and operational cost for microbial cultivation in conventional stirred tank bioreactor which makes production economically unfeasible. Non-food feedstocks, lignocellulose biomass and different waste streams containing lignocellulose, are low-cost sources of renewable carbon that could significantly reduce the production cost of microbial lipids. This review analyses the current production of microbial lipids from lignocellulose feedstocks and gives an overview of the main stages in the process of lipid production, pretreatment and hydrolysis of the feedstock and microbial cultivation. Cultivation of oleaginous microorganisms has been conducted by submerged cultivation and solid state fermentation. Three process configurations have been used in the lipid production including, separate hydrolysis and lipid production (SHLP), simultaneous saccharification and lipid production (SSLP) and consolidate bioprocessing (CBP). Implementing the biorefinery concept that includes co-production of different value-added products (polyunsaturated fatty acids, amino acids, lignin and pigments) could improve the feasibility of lipid production bioprocess

Dynamics Analysis and Biomass Productivity Optimisation of a Microbial Cultivation Process through Substrate Regulation

A microbial cultivation process model with variable biomass yield, control of substrate concentration, and biomass recycle is formulated, where the biochemical kinetics follows an extension of the Monod and Contois models. Control of substrate concentration allows for indirect monitoring of biomass and dissolved oxygen concentrations and consequently obtaining high yield and productivity of biomass. Dynamics analysis of the proposed model is carried out and the existence of order-1 periodic solution is deduced with a formulation of the period, which provides a theoretical possibility to convert the state-dependent control to a periodic one while keeping the dynamics unchanged. Moreover, the stability of the order-1 periodic solution is verified by a geometric method. The stability ensures a certain robustness of the adopted control; that is, even with an inaccurately detected substrate concentration or a deviation, the system will be always stable at the order-1 periodic solution under the control. The simulations are carried out to complement the theoretical results and optimisation of the biomass productivity is presented

High throughput Single-cell Cultivation on Microfluidic Streak Plates

This paper describes the microfluidic streak plate (MSP), a facile method for high-throughput microbial cell separation and cultivation in nanoliter sessile droplets. The MSP method builds upon the conventional streak plate technique by using microfluidic devices to generate nanoliter droplets that can be streaked manually or robotically onto petri dishes prefilled with carrier oil for cultivation of single cells. In addition, chemical gradients could be encoded in the droplet array for comprehensive dose-response analysis. The MSP method was validated by using single-cell isolation of Escherichia coli and antimicrobial susceptibility testing of Pseudomonas aeruginosa PAO1. The robustness of the MSP work flow was demonstrated by cultivating a soil community that degrades polycyclic aromatic hydrocarbons. Cultivation in droplets enabled detection of the richest species diversity with better coverage of rare species. Moreover, isolation and cultivation of bacterial strains by MSP led to the discovery of several species with high degradation efficiency, including four Mycobacterium isolates and a previously unknown fluoranthene-degrading Blastococcus species