Autotrophic Production of the Sesquiterpene α-Humulene with Cupriavidus necator in a Controlled Bioreactor.

Cupriavidus necator is a facultative chemolithotrophic organism that grows under both heterotrophic and autotrophic conditions. It is becoming increasingly important due to its ability to convert CO2 into industrially valuable chemicals. To translate the potential of C. necator into technical applications, it is necessary to optimize and scale up production processes. A previous proof-of-principle study showed that C. necator can be used for the de novo production of the terpene α-humulene from CO2 up to concentrations of 11 mg L-1 in septum flasks. However, an increase in final product titer and space-time yield will be necessary to establish an economically viable industrial process. To ensure optimized growth and production conditions, the application of an improved process design in a gas bioreactor with the control of pH, dissolved oxygen and temperature including a controlled gas supply was investigated. In the controlled gas bioreactor, the concentration of α-humulene was improved by a factor of 6.6 and the space-time yield was improved by a factor of 13.2. These results represent an important step toward the autotrophic production of high-value chemicals from CO2. In addition, the in situ product removal of α-humulene was investigated and important indications of the critical logP value were obtained, which was in the range of 3.0-4.2.

Accelerostat study in conventional and microfluidic bioreactors to assess the key role of residual glucose in the dimorphic transition of Yarrowia lipolytica in response to environmental stimuli.

Yarrowia lipolytica, with a diverse array of biotechnological applications, is able to grow as ovoid yeasts or filamentous hyphae depending on environmental conditions. This study has explored the relationship between residual glucose levels and dimorphism in Y. lipolytica. Under pH stress conditions, the morphological and physiological characteristics of the yeast were examined during well-controlled accelerostat cultures using both a 1 L-laboratory scale and a 1 mL-microfluidic bioreactor. The accelerostat mode, via a smooth increase of dilution rate (D), enabled the cell growth rate to increase gradually up to the cell wash-out (D ≥µmax of the strain), which was accompanied by a progressive increase in residual glucose concentration. The results showed that Y. lipolytica maintained an ovoid morphology when residual glucose concentration was below a threshold value of around 0.35-0.37 mg L-1. Transitions towards more elongated forms were triggered at this threshold and progressively intensified with the increase in residual glucose levels. The effect of cAMP on the dimorphic transition was assessed by the exogenous addition of cAMP and the quantification of its intracellular levels during the accelerostat. cAMP has been reported to be an important mediator of environmental stimuli that inhibit filamentous growth in Y. lipolytica by activating the cAMP-PKA regulatory pathway. It was confirmed that the exogenous addition of cAMP inhibited the mycelial morphology of Y. lipolytica, even with glucose concentrations exceeding the threshold level. The results suggest that dimorphic responses in Y. lipolytica are regulated by sugar signaling pathways, most likely via the cAMP-PKA dependent pathway.

Isopropanol production from carbon dioxide in Cupriavidus necator in a pressurized bioreactor.

A bioreactor was designed to provide high gas mass transfer to reach cell and product titres in the g L-1 level from CO2 for realistic, laboratory scale, engineered autotrophic strain evaluation. The design was based on independent CO2, H2 and air inputs and the ability to operate at high pressures. The bioreactor configuration and cultivation strategy enabled growth of Cupriavidus necator strains for long periods, to reach over 3 g L-1 dry cell weight. No negative impact of the high pressure was observed on viability of the strains up to more than 4 bar overpressure. The cultivation was then carried out using an engineered isopropanol producing strain; in this case, 3.5 g L-1 isopropanol was obtained from CO2 as the sole carbon source. This is the first reported demonstration of a successful production from engineered bacteria of product in the g L-1 range on CO2, raising the prospect of future development of CO2-based bioprocesses.

Over expression of GroESL in Cupriavidus necator for heterotrophic and autotrophic isopropanol production.

We previously reported a metabolic engineering strategy to develop an isopropanol producing strain of Cupriavidus necator leading to production of 3.4gL-1 isopropanol. In order to reach higher titers, isopropanol toxicity to the cells has to be considered. A toxic effect of isopropanol on the growth of C. necator has been indeed observed above a critical value of 15gL-1. GroESL chaperones were first searched and identified in the genome of C. necator. Native groEL and groES genes from C. necator were over-expressed in a strain deleted for PHA synthesis. We demonstrated that over-expressing groESL genes led to a better tolerance of the strain towards exogenous isopropanol. GroESL genes were then over-expressed within the best engineered isopropanol producing strain. A final isopropanol concentration of 9.8gL-1 was achieved in fed-batch culture on fructose as the sole carbon source (equivalent to 16gL-1 after taking into account evaporation). Cell viability was slightly improved by the chaperone over-expression, particularly at the end of the fermentation when the isopropanol concentration was the highest. Moreover, the strain over-expressing the chaperones showed higher enzyme activity levels of the 2 heterologous enzymes (acetoacetate carboxylase and alcohol dehydrogenase) of the isopropanol synthetic operon, translating to a higher specific production rate of isopropanol at the expense of the specific production rate of acetone. Over-expressing the native chaperones led to a 9-18% increase in the isopropanol yield on fructose.

Metabolic engineering of Cupriavidus necator for heterotrophic and autotrophic alka(e)ne production.

Alkanes of defined carbon chain lengths can serve as alternatives to petroleum-based fuels. Recently, microbial pathways of alkane biosynthesis have been identified and enabled the production of alkanes in non-native producing microorganisms using metabolic engineering strategies. The chemoautotrophic bacterium Cupriavidus necator has great potential for producing chemicals from CO2: it is known to have one of the highest growth rate among natural autotrophic bacteria and under nutrient imbalance it directs most of its carbon flux to the synthesis of the acetyl-CoA derived polymer, polyhydroxybutyrate (PHB), (up to 80% of intracellular content). Alkane synthesis pathway from Synechococcus elongatus (2 genes coding an acyl-ACP reductase and an aldehyde deformylating oxygenase) was heterologously expressed in a C. necator mutant strain deficient in the PHB synthesis pathway. Under heterotrophic condition on fructose we showed that under nitrogen limitation, in presence of an organic phase (decane), the strain produced up to 670mg/L total hydrocarbons containing 435mg/l of alkanes consisting of 286mg/l of pentadecane, 131mg/l of heptadecene, 18mg/l of heptadecane, and 236mg/l of hexadecanal. We report here the highest level of alka(e)nes production by an engineered C. necator to date. We also demonstrated the first reported alka(e)nes production by a non-native alkane producer from CO2 as the sole carbon source.

In situ rheometry of concentrated cellulose fibre suspensions and relationships with enzymatic hydrolysis.

This work combines physical and biochemical analyses to scrutinize liquefaction and saccharification of complex lignocellulose materials. A multilevel analysis (macroscopic: rheology, microscopic: particle size and morphology and molecular: sugar product) was conducted at the lab-scale with three matrices: microcrystalline cellulose (MCC), Whatman paper (WP) and extruded paper-pulp (PP). A methodology to determine on-line viscosity is proposed and validated using the concept of Metzner and Otto (1957) and Rieger and Novak's (1973). The substrate suspensions exhibited a shear-thinning behaviour with respect to the power law. A structured rheological model was established to account for the suspension viscosity as a function of shear rate and substrate concentration. The critical volume fractions indicate the transition between diluted, semi-diluted and concentrated regimes. The enzymatic hydrolysis was performed with various solid contents: MCC 273.6 gdm/L, WP 56.0 gdm/L, PP 35.1 gdm/L. During hydrolysis, the suspension viscosity decreased rapidly. The fibre diameter decreased two fold within 2 h of starting hydrolysis whereas limited bioconversion was obtained (10-15%).

Assessment of Candida shehatae viability by flow cytometry and fluorescent probes.

Quantification of different physiological states of Candida shehatae cells was performed by flow cytometry associated with two fluorescent probes. Propidium iodide and carboxyfluorescein diacetate acetoxymethyl ester fluorescent dyes were chosen based on data from the literature. A staining procedure, developed from the previous works was applied to the yeast. Then, the protocol was improved to fit with fermentation constraints such as no physiological interference between the staining procedure and the cells, shortest preparation time and small amounts of dyes. From this optimisation, propidium iodide was included in the sample at 8 mg/L whereas carboxyfluorescein was first diluted in Pluronic® agent and used at 3mg/L, samples were incubated for 10 min at 40°C. Repeatability and accuracy were evaluated to validate this flow cytometry procedure for viability determination.