The production of l-lysine by fermentation

Over 40 000 tons of L-lysine are used each year, chiefly as a nutritional supplement in animal feeds. Two major biotechnological processes are used to manufacture lysine: the enzymatic conversion of dl-α-amino-ε-caprolactam, and the direct fermentation of molasses or starch by microbes, especially species of Brevibacteria and Corynebacterium glutamicum. This review focuses on the fermentative processes and describes the development of various mutants which over-produce l-lysine.     

Stimulatory effect of glycine betaine on l-lysine fermentation

Summary The growth rate, sugar consumption rate, and production rate of an l-lysine producing Brevibacterium lactofermentum mutant were stimulated by addition of exogenous glycine betaine. Glycine betaine stimulated the growth rate especially in media of inhibitory osmotic stress, and the stimulation was independent of any specific solute. Therefore growth stimulation by glycine betaine was considered to be an osmoprotective effect. A strong enhancement of the sugar consumption rate and the l-lysine production rate was observed even with resting cells under osmotic stress as well as in a fermentation with growing cells. These data indicated that the osmoprotective effects of glycine betaine on l-lysine production can be independent of protein synthesis. Offprint requests to: Yoshio Kawahara     

Comprehensive assessment of the l-lysine production process from fermentation of sugarcane molasses

l-Lysine is an essential amino acid that can be produced by chemical processes from fossil raw materials, as well as by microbial fermentation, the latter being a more efficient and environmentally friendly procedure. In this work, the production process of l-lysine-HCl is studied using a systematic approach based on modeling and simulation, which supports decision making in the early stage of process design. The study considers two analysis stages: first, the dynamic analysis of the fermentation reactor, where the conversion of sugars from sugarcane molasses to l-lysine with a strain of Corynebacterium glutamicum is carried out. In this stage, the operation mode (either batch or fed batch) and operating conditions of the fermentation reactor are defined to reach the maximum technical criteria. Afterwards, the second analysis stage relates to the industrial production process of l-lysine-HCl, where the fermentation reactor, upstream processing, and downstream processing are included. In this stage, the influence of key parameters on the overall process performance is scrutinized through the evaluation of several technical, economic, and environmental criteria, to determine a profitable and sustainable design of the l-lysine production process. The main results show how the operating conditions, process design, and selection of evaluation criteria can influence in the conceptual design. The best plant design shows maximum product yield (0.31 g l-lysine/g glucose) and productivity (1.99 g/L/h), achieving 26.5% return on investment (ROI) with a payback period (PBP) of 3.8 years, decreasing water and energy consumption, and with a low potential environmental impact (PEI) index.

     

l-Lysine production in continuous culture of an l-lysine hyperproducing mutant of Corynebacterium glutamicum

Summary l-Lysine production was studied in and volumetric productivity were 105 g/l and 5.6 g/l per hour, respectively. The latter corresponded to a 2.5-fold higher value than that of a fed-batch culture. Higher agitation or use of oxygen-enriched air was critical for high productivity. This indicates that oxygen supply greatly affects l-lysine productivity in continuous culture.     

Corynebacterium glutamicum: morphological and ultrastructural changes of l-lysine producing cells in continuous culture

Summary Corynebacterium glutamicum was observed to undergo several morphological and ultrastructural changes during a shift in dilution rate when grown in phosphate-limiting continuous culture. At 0.1 h^–1 the cell culture appeared homogeneous and the average diameter of cells and the cell length was approximately 0.7 m and 1–1.5 m respectively. At 0.04 h^–1 there was essentially no change in these readings, but at this dilution rate there was a significant proportion of cells that measured three times the original length. At six residence times the elongated cells were increasing in number, but these changes occurred without diminishing the system's performance. Some changes in cell ultrastructure were observed during the shift in dilution rate. Thus, the presence of polyphosphate granules and of glycogen appeared in the cytoplasm of producing cells (0.04 h^–1). We have confirmed by mass spectrometry the absence of poly--hydroxybutyrate (PHB) in C. glutamicum.Correspondence to: N. Coello     

Modelling of continuous acetonobutylic fermentation

A model of continuous acetonobutylic fermentation is proposed. This model correctly portrays the predominantly solvents formation observed at acidic extracellular pH and predominantly acids production at more neutral pH, as well as observed effects of dilution rate and feed substrate on products' concentrations. A fair agreement between experimental and theoretical predictions is achieved for a broad range of operating variables.     

Ethanol fermentation in a continuous tower fermentor

A mutant of Saccharomyces cerevisiae, which forms large, multicellular flocs in liquid culture, rapidly fermented media containing high concentrations of glucose (100-180 g/L) in a continuous nonaerated tower fermentor at 30 degrees C. The fermentor operated continuously for seven months. Batch and tower fermentor data were fitted to a kinetic model incorporating linear ethanol inhibition and Monod dependence on glucose. Conversion, ethanol yield, and ethanol productivity were related to the apparent fermentation time for initial glucose concentrations of 130 and 180 g/L. Productivities of 8-12 g ethanol/L h were achieved through the yeast bed giving conversions exceeding 90% of the theoretical yield.     

Xanthan production byXanthomonas campestris in continuous fermentation

Summary Xanthan production by a strain ofX. campestris was maintained longer under glucose—limited than nitrogen—limited conditions in continuous culture. The turnover Q was 12 at lower pH(5.0–6.0) and 6.5 at neutral pH; xanthan productivity was comparable at both pH in nitrogen—limited continuous cultures. In the Fe—rich continuous fermentation cell degeneration did not occur for 65 turnovers whereas in the Fe—deficient fermentation it occurred in 12 turnovers. The culture stability for xanthan formation is higher under conditions which are favorable for cell growth with limited xanthan production.     

Characterisation of kinetic parameters and metabolic transition of Corynebacterium glutamicum on l-lysine production in continuous culture

Kinetic parameters and physiological states of Corynebacterium glutamicum at the growing and l-lysine-overproducing phase were characterised in continuous culture on threonine-limited complex and minimal media. High l-lysine productivity occurred at dilution rates ranging from 0.1 h^–1 to 0.3 h^–1 on threonine-limited complex medium, and at dilution rates ranging from 0.1 h^–1 to 0.15 h^–1 in minimal medium. l-Lysine yields of 0.25 g/g (0.31 g/g as l-lysine hydrochloride) in complex medium, and of 0.17 g/g (0.21 g/g as l-lysine hydrochloride) in minimal medium, corresponding respectively to intrinsic yields of 0.533 g/g and 0.572 g/g were obtained. These intrinsic yield factors are closed to the theoretical ones (0.608 g/g, 0.75 mol/mol). Intrinsic biomass yields were calculated as 0.658 g/g in complex medium and 0.283 g/g in minimal medium. CO₂ production has been clearly related to l-lysine production. According to our results on specific uptake rates and specific productivities in complex medium, metabolic rearrangement should occur during the transition from the growing phase to the l-lysine-overproducing phase. This phenomenon was further investigated.     

Optimal control of continuous fermentation processes

Three layer control structure is proposed for optimal control of continuous fermentation processes. The start-up optimization problems are solved as a first step for optimization layer building. A steady state optimization problem is solved by a decomposition method using prediction principle. A discrete minimum time optimal control problem with state delay is formulated and a decomposition method, based on an augmented Lagrange's function is proposed to solve it. The problem is decomposed in time domain by a new coordinating vector. The obtained algorithms are used for minimum time optimal control calculation of Baker's Yeast fermentation process.List of Symbols x(t) g/l biomass concentration - s(t) g/l limiting substrate concentration - x ⁰ g/l inlet biomass concentration - s ⁰(t) g/l inlet substrate concentration - D(t) h^–1 dilution rate - (t) h^–1 specific growth rate - Y g/g yield coefficient - (t) h^–1 specific limiting substrate consumption rate - k _D h^–1 disappearing constant - w ₁, w ₂ known constant or piece-wise disturbances - _m h^–1 maximum specific growth rate - k _s g/l Michaelis-Menten's parameter - h time delay - x ₀, s ₀ g/l initial concentrations - ¯x, ¯s, ¯D optimal steady state value - V _min , V _max , v=x,s,d,t bounds of variables - t h sampling period - K number of steps in the optimization horison - Js, J _d performance indexes - L _s Lagrange's function - L _d Lagrange's functional - ₀ weighting coefficient for the amount of the limiting substrate throwing out of the fermentor - ₁, ₂ dual variables of Lagrange's function - steps in steady state coordination procedure - errors values for steady state coordination process - _v , v=x, s conjugate variables of Lagrange's functional - _v , v=x,s penalty coefficients of augmented Lagrange's functional - _v , v=x, s interconnections of the time - e _v , v=x,s, D, _x , _s gradients of Lagrange's functional - j, l indexes of calculation procedures - values of errors in calculations The researches was supported by National Scientific Research Foundation under grants No NITN428/94 and No NITN440/94     

Modelling of oscillatory behaviour in continuous ethanol fermentation

Summary It is shown that a tri-compartment cell population model with kinetics predicting decrease of product inhibition with decrease in substrate concentration can portray all dynamic situations observed in continuous ethanol fermentations depending on substrate concentration, from sustained oscillations to completely stable cultures.     

Amperometric l-lysine determination biosensor amplified with l-lysine oxidase nanoparticles and graphene oxide nanoparticles

We describe the amplification of amperometric l-lysine biosensor using l-lysine oxidase nanoparticles (LOxNPs) and graphene oxide nanoparticles (GrONPs) immobilized onto pencil graphite electrode (PGE). LOxNPs and GrONPs were characterized by UV spectroscopy and transmission electron microscopy (TEM). The working electrode (LOxNPs/GrONPs/PGE) was studied by scanning electron microscopy (SEM) and cyclic voltammetry at different stages of its construction. The biosensor showed optimum current at 0.7 V, pH 6.5, 35 °C, a detection limit of 0.01 μM, response time as 3.95 s and a wider linear range 0.01–1000 μM. The analytical recovery of added lysine in sera was 97 %. The within assay and between batch coefficients of variation for the biosensor were 0.068 and 0.074 % respectively. The biosensor measured l-lysine levels in sera of healthy adults and human immunodeficiency virus (HIV) patients. The biosensor exhibited good correlation with standard spectrophotometric method (R² = 0.989). The biosensor lost 35 % of its original activity after its regular uses for a period of 180 days, while being stored dry at 4 °C.     

On the controllability of continuous fermentation processes

Simulation may be used as a powerful tool for accelerating bioprocess design. This paper demonstrates the use of simulations in exploring the nature and impact of the interactions that exist in a typical bioprocess for the recovery of an intracellular protein. The study shows that an integrated approach to design must be adopted in order to achieve acceptable process designs. Data from a fed-batch fermentation, with verified models for cell harvesting, cell disruption and cell debris removal have been integrated to demonstrate the consequence of process design and operating decisions on the resulting process performance. The trade-offs between product recovery and the extent of cell debris removal for a range of operating conditions have been represented through a series of windows of operation which show how process conditions must be altered in order for given process performance levels to be realised. The capacity to account for process performance including the impact of interactions is seen as a pre-requisite for rigorous bioprocess sequence design and optimisation.     

Morphological behaviour ofSaccharomyces cerevisiae during continuous fermentation

Summary Saccharomyces cerevisiae were observed to undergo drastic morphological changes when grown in continuous culture. Peak elongations and minimum cell volumes were found at intermediate dilution rates when it was believed the insitu glucose concentration was at its lowest. The shapes and sizes were reproducible and have been quantified at two different glucose feed concentrations.     

Approximation of continuous fermentation by semicontinuous cultures

Semicontinuous fermentations, in which a fraction of a culture is replaced with fresh media at regular intervals, have been previously used as a means of approximating continuous growth. In most cases deviations from continuous operation were erroneously estimated using Fencl's model, which is only valid when the specific growth rate is independent of the substrate concentration. An approach to modeling Semicontinuous growth that incorporates the same kinetics followed in batch and continuous growth was developed and tested for Monod's expression for the specific growth rate. A dimensionless form of the model was used to simulate Semicontinuous fermentations for comparison to continuous growth. Differences between Semicontinuous and continuous growth were found to depend on three dimensionless variables: feed concentration, replacement rate, and time between replacements. For given values of the dimensionless feed concentration and time between replacements, a range of dimensionless replacement rates can be determined over which semi-continuous cultures are approximately continuous.     

Robust compensator control of continuous fermentation processes

The paper deals with the robust compensator control of continuous fermentation processes described by a set of three non-linear differential equations. For the design purposes the non-linear model is transformed into linear one with interval parameters. Robust state space compensator is designed by the internal model principle, which ensures robust step-wise set points asymptotic tracking and external disturbances rejection in the wide working range. The effectiveness of the algorithm designed is performed by computer simulation experiments. An important feature of the proposed algorithms is their robustness over parameter uncertainties in the process models.