Acetone production with metabolically engineered strains of Acetobacterium woodii.

Expected depletion of oil and fossil resources urges the development of new alternative routes for the production of bulk chemicals and fuels beyond petroleum resources. In this study, the clostridial acetone pathway was used for the formation of acetone in the acetogenic bacterium Acetobacterium woodii. The acetone production operon (APO) containing the genes thlA (encoding thiolase A), ctfA/ctfB (encoding CoA transferase), and adc (encoding acetoacetate decarboxylase) from Clostridium acetobutylicum were cloned under the control of the thlA promoter into four vectors having different replicons for Gram-positives (pIP404, pBP1, pCB102, and pCD6). Stable replication was observed for all constructs. A. woodii [pJIR_actthlA] achieved the maximal acetone concentration under autotrophic conditions (15.2±3.4mM). Promoter sequences of the genes ackA from A. woodii and pta-ack from C. ljungdahlii were determined by primer extension (PEX) and cloned upstream of the APO. The highest acetone production in recombinant A. woodii cells was achieved using the promoters PthlA and Ppta-ack. Batch fermentations using A. woodii [pMTL84151_actthlA] in a bioreactor revealed that acetate concentration had an effect on the acetone production, due to the high Km value of the CoA transferase. In order to establish consistent acetate concentration within the bioreactor and to increase biomass, a continuous fermentation process for A. woodii was developed. Thus, acetone productivity of the strain A. woodii [pMTL84151_actthlA] was increased from 1.2mgL(-1)h(-1) in bottle fermentation to 26.4mgL(-1)h(-1) in continuous gas fermentation.

Implementation of Perforated Concentric Ring Walls Considerably Improves Gas-Liquid Mass Transfer of Shaken Bioreactors.

Since their first use in the 1930s, shake flasks have been a widely used bioreactor type for screening and process development due to a number of advantages. However, the limited gas-liquid mass transfer capacities-resulting from practical operation limits regarding shaking frequency and filling volumes-are a major drawback. The common way to increase the gas-liquid mass transfer in shake flasks with the implementation of baffles is generally not recommended as it comes along with several severe disadvantages. Thus, a new design principle for shaken bioreactors that aims for improving the gas-liquid mass transfer without losing the positive characteristics of unbaffled shake flasks is introduced. The flasks consist of cylindrical glass vessels with implemented perforated concentric ring walls. The ring walls improve the gas-liquid mass transfer via the formation of additional liquid films on both of its sides, whereas the perforations allow for mixing between the compartments. Sulfite oxidation experiments revealed over 200% higher maximum oxygen transfer capacities (OTRmax) compared to conventional shake flasks. In batch cultivations of Escherichia coli BL21 in mineral media, unlimited growth until glucose depletion and oxygen transfer rates (OTR) of up to 138 mmol/L/h instead of an oxygen limitation at 57 mmol/L/h as in normal shake flasks under comparable conditions could be achieved. Even overflow metabolism could be prevented due to sufficient oxygen supply without the use of unconventional shaking conditions or oxygen enrichment. Therefore, we believe that the new perforated ring flask principle has a high potential to considerably improve biotechnological screening and process development steps.