ABSTRACT
The two major scale-up criteria in continuously stirred bioreactors are 1) constant aerated power input per volume (Pg/Vl), and 2) the volumetric O2 mass transfer coefficient (kla). However, Pg/Vl is only influenced by the stirrer geometry, stirrer speed, aeration and working volume, while the kla is additionally affected by physiochemical properties of the medium (temperature, pH, salt content, etc.), sparging of gas and also by the bioreactor design. The extremophilic archaeon Sulfolobus acidocaldarius, thriving at 75°C and pH 3.0, has the potential for many biotechnological applications. However, previous studies imply that the family Sulfolobaceae might be affected by higher oxygen concentration in the headspace (>26%). Hence, adequate oxygen supply without being toxic has to be ensured throughout the different scales. In this study, the scale-up criteria Pg/Vl and kla were analyzed and compared in a 2 L chemostat cultivation of S. acidocaldarius on a defined growth medium at 75°C and a pH value of 3.0, using two different types of spargers at the same aerated power input. The scale-up criterion kLa, ensuring a high specific growth rate as well as viability, was then used for scaleup to 20 L and 200 L. By maintaining a constant kla comparable dry cell weight, specific growth rate, specific substrate uptake rates and viability were observed between all investigated scales. This procedure harbors the potential for further scale-up to industrial size bioreactors.
ABSTRACT
Determination of the viability, ratio of dead and live cell populations, of Sulfolobus acidocaldarius is still being done by tedious and material-intensive plating assays that can only provide time-lagged results. Although S. acidocaldarius, an extremophilic Archaeon thriving at 75 °C and pH 3.0, and related species harbor great potential for the exploitation as production hosts and biocatalysts in biotechnological applications, no industrial processes have been established yet. One hindrance is that during development and scaling of industrial bioprocesses timely monitoring of the impact of process parameters on the cultivated organism is crucial-a task that cannot be fulfilled by traditional plating assays. As alternative, flow cytometry (FCM) promises a fast and reliable method for viability assessment via the use of fluorescent dyes. In this study, commercially available fluorescent dyes applicable in S. acidocaldarius were identified. The dyes, fluorescein diacetate and concanavalin A conjugated with rhodamine, were discovered to be suitable for viability determination via FCM. For showing the applicability of the developed at-line tool for bioprocess monitoring, a chemostat cultivation on a defined growth medium at 75 °C, pH 3.0 was conducted. Over the timeframe of 800 h, this developed FCM method was compared to the plating assay by monitoring the change in viability upon controlled pH shifts. Both methods detected an impact on the viability at pH values of 2.0 and 1.5 when compared to pH 3.0. A logarithmic relationship between the viability observed via plating assay and via FCM was observed.
ABSTRACT
The crenarchaeal model organism Sulfolobus acidocaldarius is typically cultivated in shake flasks. Although shake flasks represent the state-of-the-art for the cultivation of this microorganism, in these systems crucial process parameters, like pH or substrate availability, are only set initially, but cannot be controlled during the cultivation process. As a result, a thorough characterization of growth parameters under controlled conditions is still missing for S. acidocaldarius. In this study, we conducted chemostat cultivations at 75 °C using a growth medium containing L-glutamate and D-glucose as main carbon sources. Different pH values and dilution rates were applied with the goal to physiologically characterize the organism in a controlled bioreactor environment. Under these controlled conditions a pH optimum of 3.0 was determined. Washout of the cells occurred at a dilution rate of 0.097 h-1 and the optimal productivity of biomass was observed at a dilution rate of 0.062 h-1. While both carbon sources were taken up by S. acidocaldarius concomitantly, a 6.6-fold higher affinity for L-glutamate was shown. When exposed to suboptimal growth conditions, S. acidocaldarius reacted with a change in the respiratory behavior and an increased trehalose production rate in addition to a decreased growth rate.
Subject(s)
Sulfolobus acidocaldarius , Biomass , Bioreactors , Culture Media , GlucoseABSTRACT
The microbial cell membrane is affected by physicochemical parameters, such as temperature and pH, but also by the specific growth rate of the host organism. Homeoviscous adaption describes the process of maintaining membrane fluidity and permeability throughout these environmental changes. Archaea, and thereby, Sulfolobus spp. exhibit a unique lipid composition of ether lipids, which are altered in regard to the ratio of diether to tetraether lipids, number of cyclopentane rings and type of head groups, as a coping mechanism against environmental changes. The main biotechnological application of the membrane lipids of Sulfolobus spp. are so called archaeosomes. Archaeosomes are liposomes which are fully or partly generated from archaeal lipids and harbor the potential to be used as drug delivery systems for vaccines, proteins, peptides and nucleic acids. This review summarizes the influence of environmental parameters on the cell membrane of Sulfolobus spp. and the biotechnological applications of their membrane lipids.
