Quasi steady state growth of Lactococcus lactis in glucose-limited acceleration stat (A-stat) cultures

Abstract

Quasi steady state growth of Lactococcus lactis IL 1403 was studied in glucose-limited A-stat cultivation experiments with acceleration rates (a) from 0.003 to 0.06 h(-2) after initial stabilization of the cultures in chemostat at D = 0.2-0.3 h(-1). It was shown that the high limit of quasi steady state growth rate depended on the acceleration rate used-at an acceleration rate 0.003 h(-2) the quasi steady state growth was observed until mu (crit) = 0.59 h(-1), which is also the mu (max) value for the culture. Lower values of mu (crit) were observed at higher acceleration rates. The steady state growth of bacteria stabilized at dilution rate 0.2 h(-1) was immediately disrupted after initiating acceleration at the highest acceleration rate studied-0.06 h(-2). Observation was made that differences [Delta(mu - D)] of the specific growth rates from pre-programmed dilution rates were the lowest using an acceleration rate of 0.003 h(-2) (< 4% of preset changing growth rate). The adaptability of cells to follow preprogrammed growth rate was found to decrease with increasing dilution rate-it was shown that lower acceleration rates should be applied at higher growth rates to maintain the culture in the quasi steady state. The critical specific growth rate and the biomass yields based on glucose consumption were higher if the medium contained S (0) = 5 g L(-1) glucose instead of S (0) = 10 g L(-1). It was assumed that this was due to the inhibitory effect of lactate accumulating at higher concentrations in the latter cultures. Parallel A-stat experiments at the same acceleration and dilution rates showed good reproducibility-Delta(mu - D) was less than 5%, standard deviations of biomass yields per ATP produced (Y (ATP)), and biomass yields per glucose consumed (Y (XS)) were less than 15%.

Advanced continuous cultivation methods for systems microbiology

“…This is supported by several studies where m max was reached for different micro-organisms in A-stats (Adamberg et al, 2009;Barbosa et al, 2003;Kask et al, 1999;Paalme et al, 1995Paalme et al, , 1997aValgepea et al, 2010). A thorough study of the effect of acceleration on the growth of L. lactis showed that m max of 0.59 h 21 was reached in an A-stat whilst maintaining the quasi steady state using accelerations v0.005 h 22 (Adamberg et al, 2009). Another study concluded that an acceleration of 0.001 h 22 is the fastest that can be used for approaching steady-state culture characteristics during A-stats with yeasts (van der Sluis et al, 2001).…”

“…For an A-stat experiment, appropriate acceleration can be estimated based on m max of the organism to be in the range of 0.01-0.04|m max (Kasemets et al, 2003). This is supported by several studies where m max was reached for different micro-organisms in A-stats (Adamberg et al, 2009;Barbosa et al, 2003;Kask et al, 1999;Paalme et al, 1995Paalme et al, , 1997aValgepea et al, 2010). A thorough study of the effect of acceleration on the growth of L. lactis showed that m max of 0.59 h 21 was reached in an A-stat whilst maintaining the quasi steady state using accelerations v0.005 h 22 (Adamberg et al, 2009).…”

“…) resulting in hysteresis of metabolism: activation of arginine degradation with a decrease of pH to 5.4 was not deactivated at the end of the experiment after pH had been increased back to the initial pH of 6.3 (Lahtvee et al, 2009). Additionally, a very long changestat experiment due to slow acceleration risks the of emergence of unwanted mutations in the cell culture, similar to what is seen in chemostats (Ferenci, 2007;Gresham & Hong, 2015;Harder et al, 1977;Helling et al, 1987;Novick & Szilard, 1950).…”

“…The same study also utilized a D-stat to determine the metabolic events accompanying the transition of metabolism from carbon to nitrogen limitation in S. cerevisiae by smoothly decreasing the nitrogen/carbon ratio in the ingoing medium. Similarly, analysis of the effect(s) of pH and/or temperature on L. lactis growth physiology showed dependence of both biomass yield and lactate production on pH and temperature (Lahtvee et al, 2009). Additionally, substrate consumption and product formation patterns, and gene expression showed no difference between increasing or decreasing pH within the range of 5.4-6.3.…”

The modeling of ethanol production by Kluyveromyces marxianus using whey as substrate in continuous A-Stat bioreactors

“…This behavior showed great influence in the value of the yield coefficient for product on substrate (Y P/S ), which reached 0.40 g g −1 for the highest acceleration rate, and 0.52 g g −1 for the lowest acceleration rate. The effect of acceleration rates on the specific growth rate was reported in other researches, showing that lower acceleration rates lead to the approximation of steady state [1,3,22,50]. Therefore, it is clear that the choice of acceleration rate is a critical step in A-stat systems.…”