Catherine Creuly

Pyrazine production by Bacillus subtilis in solid-state fermentation on soybeans

Abstract 2,5-Dimethylpyrazine (2,5-DMP) and tetramethylpyrazine (TTMP) were produced using Bacillus subtilis IFO 3013 grown on soybeans. Solid-state cultivations were carried out either in 100-ml bottles or in a fixed-bed column reactor, both systems being at 27 °C. Optimization studies showed that the best way to produce the two above aroma compounds involved two separate processes. 2,5-DMP was obtained using soybeans enriched with 75 g threonine/kg initial dry weight (i.d.w.), giving 0.85 g metabolite/kg i.d.w. after 6 days. TTMP production involved addition of 90 g/kg i.d.w. acetoin to soybeans, and 2.5 g/kg i.d.w. was recovered after 14 days. These results demonstrated the suitability of solid-state cultivation for production of high-added-value compounds.

Advanced Anaerobic Bioconversion of Lignocellulosic Waste for Bioregenerative Life Support following Thermal Water Treatment and Biodegradation by Fibrobacter Succinogenes

The feasibility of nearly-complete conversion of lignocellulosic waste (70% food crops, 20% faecal matter and 10% green algae) into biogas was investigated in the context of a life support project. The treatment comprised a series of processes, i.e., a mesophilic laboratory scale CSTR (continuously stirred tank reactor), an upflow biofilm reactor, a fiber liquefaction reactor employing the rumen bacterium Fibrobacter succinogenes and a hydrothermolysis system in near-critical water. By the one-stage CSTR, a biogas yield of 75% with a specific biogas production of 0.37 l biogas g−1 VSS (volatile suspended solids) added at a RT (hydraulic retention time) of 20–25 d was obtained. Biogas yields could not be increased considerably at higher RT, indicating the depletion of readily available substrate after 25 d. The solids present in the CSTR-effluent were subsequently treated in two ways. Hydrothermal treatment (T ∼ 310–350 °C, p ∼ 240 bar) resulted in effective carbon liquefaction (50–60% without and 83% with carbon dioxide saturation) and complete sanitation of the residue. Application of the cellulolytic Fibrobacter succinogenes converted remaining cellulose contained in the CSTR-effluent into acetate and propionate mainly. Subsequent anaerobic digestion of the hydrothermolysis and the Fibrobacter hydrolysates allowed conversion of 48–60% and 30%, respectively. Thus, the total process yielded biogas corresponding with conversions up to 90% of the original organic matter. It appears that particularly mesophilic digestionin conjunction with hydrothermolysis at near-critical conditions offers interesting features for (nearly) complete and hygienic carbon and energy recovery from human waste in a bioregenerative life support context.

Bioconversion of fatty acids into methyl ketones by spores of Penicillium roquefortii in a water-organic solvent, two-phase system

Abstract C 5 –C 9 methyl ketones can be produced from the corresponding C 6 –C 10 fatty acids by spores of Penicillium roquefortii in a water—organic solvent, two-phase system operated using a fed-batch procedure carried out at constant substrate concentration. The organic phase consists of an industrial isoparaffin solvent which can be considered as tetradecane. The reaction is initially performed by resting spores, which have the best biocatalyst activity, and swelling of spores appears later. Optimization involves both increased initial reaction rate and late spore swelling. The biocatalyst is obtained by cultivation of the fungus by solid state fermentation on buckwheat seeds, and the simplest way to carry out bioconversion reactions is to use the whole sporulation medium without discarding the buckwheat grains. This method gives the best results, and 21 g l −1 2-pentanone, 73 g l −1 2-heptanone, and 57 g l −1 2-nonanone were recovered in this way.

A fed-batch technique for 2-heptanone production by spores of Penicillium roquefortii.

SummaryA fed-batch technique for the production of 2-heptanone from octanoic acid by Ca-alginate/Eudragit RL-entrapped spores of Penicillium roquefortii is presented. It involves the use of a pH-stat apparatus, the increase in pH arising due to the reaction being overcome by direct substrate addition. When the reaction is performed at pH 5.5, no true constant rate period can be achieved due to a gradual buffering of the medium connected with a decrease in the catalytic activity of the spores. At pH 6.5 the system exhibits high stability and the reaction rate remains constant for at least 300 h with a value of 0.975 mM/h and a substrate concentration close to 4 mM. The process used may be very useful in the study of long-term behaviour of biocatalysts when the medium is not renewed and when the reaction involves an ionic compound and a neutral one.

Dynamic Aspects and Controllability of the MELiSSA Project: A Bioregenerative System to Provide Life Support in Space

Manmade ecosystems differ from their prototype biosphere by the principle of control. The Earth Biosphere is sustainable by stochastic control and very large time constants. By contrast, in a closed ecosystem such as the micro-ecological life support system alternative (MELiSSA system) developed by the European Space Agency for space exploration, a deterministic control is a prerequisite of sustainable existence. MELiSSA is an integrated sum of interconnected biological subsystems. On one hand, all unit operations in charge of the elementary functions constitutive of the entire life support system are studied until a thorough understanding and mathematical modelling. On the other hand, the systemic approach of complex, highly branched systems with feedback loops is performed. This leads to study in the same perspective, with the same degree of accuracy and with the same language, waste degradation, water recycling, atmosphere revitalisation and food production systems prior to the integration of knowledge-based control models. This paper presents the mathematical modelling of the MELiSSA system and the interface between the control strategy of the entire system and the control of the bioreactors.

2-HEPTANONE PRODUCTION BY SPORES OF Penicillium roquefortii IN A WATER- ORGANIC SOLVENT TWO-PHASE SYSTEM

The production of 2-heptanone from octanoic acid may be performed by free and entrapped spores of Penicillium roquefortii in a water-organic solvent two phase system.An industrial, isoparafflnic solvent, i.e. Hydrosol IP 230 O.S., which may be considered as tetradecane, is well suited for the process. Activities nearly double those achieved with aqueous systems are observed using an initial fatty acid content in the organic layer close to 100 mM and a ratio of the volume of the organic phase to the total volume of the medium of 0.88. The presence of the solvent allows a better recovery of the metabolite by lowering its activity coefficient.Fed-batch experiments performed in an aerated, stirred reactor show that the bioconversion may proceed in the two-phase system for at least 300 h. These conditions allow conversion of 750 mM (108 g · 1-1) fatty acid, and production of 600 mM (68.5 g · 1-1) 2-heptanone.

Metabolic Flux Modelling as a Tool to Analyse the Behavior of a Genetically Modified Strain of Saccharomyces Cerevisiae

Flux distribution for a wild and a mutant strain of Saccharomyces cerevisiae are compared and investigated in terms of metabolic flux calculation and thermodynamic analysis of central metabolism under anaerobic conditions. Starting from a redundant set of measured rates obtained from batch cultures on glucose or fructose as carbon source, an original data reconciliation technique associated with the calculation of metabolic flux is used. Comparative analysis of carbon split in the metabolic network for the mutant yeast strain lacking the glucose6P-dehydrogenase (CD101-1A) and for the reference wild strain (ATCC 7754) allows to conclude that the pentose phosphate is in priority devoted to its anabolic function rather than to the production of NADPH cofactors. This last function seems to be as well assumed by the specific NADP acetaldehyde dehydrogenase enzyme; this explains the significantly higher production of acetate by the mutant strain.

Growth monitoring of Fibrobacter succinogenes by pressure measurement.

In life support systems, such as the MELiSSA (Micro-Ecological Life Support Alternative) project, developed by the European Space Agency, the aim is to understand and assemble artificial ecosystems for ensuring human subsistence in space. Fibrobacter succinogenes, an anaerobic bacterium, was used for the degradation of vegetable wastes produced in higher plants chambers, but the process does not allow the monitoring of biomass concentration and degradation rates. This study proposes a growth and a degradation monitoring technique using pressure measurements. First, volatile fatty acids (VFA) production was compared with biomass growth and with CO2 production. The experiments were carried out in batch and fed-batch processes on glucose and on vegetables. The results have shown that a link could be established between VFA production, degradation rate and gas pressure measurements. Thus, the pressure could be used both as a relevant variable for online evaluation of biomass growth and of degradation of complex vegetable wastes.

Test of an anaerobic prototype reactor coupled with a filtration unit for production of VFAs

The artificial ecosystem MELiSSA, supported by the European Space Agency is a closed loop system consisting of 5 compartments in which food, water and oxygen are produced out of organic waste. The first compartment is conceived as a thermophilic anaerobic membrane bioreactor liquefying organic waste into VFAs, ammonium and CO2 without methane. A 20 L reactor was assembled to demonstrate the selected design and process at prototype scale. We characterized system performance from start-up to steady state and evaluated process efficiencies with special attention drawn to the mass balances. An overall efficiency for organic matter biodegradation of 50% was achieved. The dry matter content was stabilized around 40-50 g L(-1) and VFA production around 5-6 g L(-1). The results were consistent for the considered substrate mixture and can also be considered relevant in a broader context, as a first processing step to produce building blocks for synthesis of primary energy vectors.

Growth modelling of Nitrosomonas europaea ATCC® 19718 and Nitrobacter winogradskyi ATCC® 25391: A new online indicator of the partial nitrification

The aim of the present work was to study the growth of two nitrifying bacteria. For modelling the nitrifying subsystem of the MELiSSA loop, Nitrosomonas europaea ATCC® 19718 and Nitrobacter winogradskyi ATCC® 25931 were grown separately and in cocultures. The kinetic parameters of a stoichiometric mass balanced Pirt model were identified: μmax=0.054h(-1), decay rate b=0.003h(-1) and maintenance rate m=0.135gN-NH4(+)·gX(-1)·h(-1) for Nitrosomonas europaea; μmax=0.024h(-1), b=0.001h(-1) and m=0.467gN-NO2(-)·gX(-1)·h(-1) for Nitrobacter winogradskyi. A predictive structured model of nitrification in co-culture was developed. The online evolution of the addition of KOH is correlated to the nitritation; the dissolved oxygen concentration is correlated to both nitritation and nitratation. The model suitably represents these two variables so that transient partial nitrification is assessed. This is a clue for avoiding partial nitrification by predictive functional control.