Laurent Poughon

Energy model and metabolic flux analysis for autotrophic nitrifiers

The behavior of pure cultures of nitrifying microorganisms under autotrophic growth operating conditions was investigated and the relations between their energy metabolism and their anabolism analyzed by means of metabolic network computation. The description of the metabolism of the nitrifiers is extended to their energy metabolism by introducing compartmentalization (cytoplasmic and periplasmic sides) and studying coupling between the electron transport chain and the proton gradient generation. The energy model of Nitrosomonas and Nitrobacter was developed based on the oxidoreduction reactions known to be involved. The electron transport chains and the associated proton translocation for these models are described. Several possible hypotheses are analyzed and discussed concerning the thermodynamic consistency of all the oxidoreduction reactions. For Nitrosomonas, the most delicate point is the second step of hydroxylamine oxidation. For Nitrobacter a new energy model is proposed in which NO plays an important role as node in the distribution of electrons from NO(2)(-) oxidation to the membrane electron transport chain. The compartmentalization enables us to consider a proton gradient dissipation flux as the expression of the overall energy loss in metabolic analysis (the so-called maintenance phenomena). The energy model (electron transport chain, proton gradient) is associated with an overall description of the metabolism of Nitrosomonas and Nitrobacter in terms of metabolic flux calculation. This representation demonstrates that a maintenance in nitrifiers expressed as a proton leak is no higher than for other aerobes. The yields calculated from the energy models integrated with the metabolic models of nitrifiers are consistent with the experimental yields in the literature.

Recycling efficiencies of C,H,O,N,S, and P elements in a biological life support system based on micro-organisms and higher plants

MELiSSA is a microorganism based artificial ecosystem conceived as a tool for understanding the behavior of ecosystems and developing the technology for future Manned Space Missions. MELiSSA is composed of four compartments colonized by the microorganisms required by the function of this ecosystem : breakdown of waste produced by men, regeneration of atmosphere and biosynthesis of edible biomass. This paper reports the mass balance description of a Biological Life Support System composed of the MELiSSA loop and of a Higher Plant Compartment working in parallel with the photosynthetic Spirulina compartment producing edible biomass. The recycling efficiencies of the system are determined and compared for various working conditions of the MELiSSA loop with or without the HPC.

Characterization of arabinogalactan-rich mucilage from Cereus triangularis cladodes

Cereus triangularis (Cactaceae) is a cactus used in food decoction as a traditional medicine in the North region of Madagascar to reduce stomach ache and intestinal diseases. Hydrocolloids were sequentially extracted from its cladodes with a yield of 24% (240 mg/g based on dried cladodes powder). Structural analyses has revealed that this polysaccharide with a molecular mass of 8430,000g/mol was mainly composed of a galactan backbone of a (1 → 4) linked β-d-Galp residues probably substituted at position 3 by L-arabinofuranosyl residues. In vitro antioxidant activity of this arabinogalactan-rich fraction was detected and quantified by radical DPPH scavenging, hydroxyl radical scavenging, radical anion superoxide scavenging and reducing power method.

Cellulase activity mapping of Trichoderma reesei cultivated in sugar mixtures under fed-batch conditions

BackgroundOn-site cellulase production using locally available lignocellulosic biomass (LCB) is essential for cost-effective production of 2nd-generation biofuels. Cellulolytic enzymes (cellulases and hemicellulases) must be produced in fed-batch mode in order to obtain high productivity and yield. To date, the impact of the sugar composition of LCB hydrolysates on cellulolytic enzyme secretion has not been thoroughly investigated in industrial conditions.ResultsThe effect of sugar mixtures (glucose, xylose, inducer) on the secretion of cellulolytic enzymes by a glucose-derepressed and cellulase-hyperproducing mutant strain of Trichoderma reesei (strain CL847) was studied using a small-scale protocol representative of the industrial conditions. Since production of cellulolytic enzymes is inducible by either lactose or cellobiose, two parallel mixture designs were performed separately. No significant difference between inducers was observed on cellulase secretion performance, probably because a common induction mechanism occurred under carbon flux limitation. The characteristics of the enzymatic cocktails did not correlate with productivity, but instead were rather dependent on the substrate composition. Increasing xylose content in the feed had the strongest impact. It decreased by 2-fold cellulase, endoglucanase, and cellobiohydrolase activities and by 4-fold β-glucosidase activity. In contrast, xylanase activity was increased 6-fold. Accordingly, simultaneous high β-glucosidase and xylanase activities in the enzymatic cocktails seemed to be incompatible. The variations in enzymatic activity were modelled and validated with four fed-batch cultures performed in bioreactors. The overall enzyme production was maintained at its highest level when substituting up to 75% of the inducer with non-inducing sugars.ConclusionsThe sugar substrate composition strongly influenced the composition of the cellulolytic cocktail secreted by T. reesei in fed-batch mode. Modelling can be used to predict cellulolytic activity based on the sugar composition of the culture-feeding solution, or to fine tune the substrate composition in order to produce a desired enzymatic cocktail.

Improvement and modeling of culture parameters to enhance biomass and lipid production by the oleaginous yeast Cryptococcus curvatus grown on acetate.

The improvement of culture parameters for lipid production from acetate as carbon source was investigated using the oleaginous yeast Cryptococcus curvatus. A new pH regulation system dispensing acetate was developed for fed-batch culture and allowed obtaining nearly 80 g/L biomass within 60 h with a maximal growth rate of 0.28 h(-1). A biological model was developed from experimental data. The influence of three C/N ratios of 300, 500 and 900 were tested during a multi-phases process on lipid accumulation. The C/N ratio of 300 was reported to be the most suitable for lipid storage. No significant increase of lipids content was obtained with higher value. A maximal content of 60% DCW of lipid was obtained. The determination of fatty acids profiles of the microbial oils has confirmed that the valorization of acetate by microbial oils production was a promising perspective.

A new stoichiometric miniaturization strategy for screening of industrial microbial strains: application to cellulase hyper-producing Trichoderma reesei strains

BackgroundDuring bioprocess development, secondary screening is a key step at the boundary between laboratory and industrial conditions. To ensure an effective high-throughput screening, miniaturized laboratory conditions must mimic industrial conditions, especially for oxygen transfer, feeding capacity and pH stabilization.ResultsA feeding strategy has been applied to develop a simple screening procedure, in which a stoichiometric study is combined with a standard miniaturization procedure. Actually, the knowledge of all nutriments and base or acid requirements leads to a great simplification of pH stabilization issue of miniaturized fed-batch cultures. Applied to cellulase production by Trichoderma reesei, this strategy resulted in a stoichiometric mixed feed of carbon and nitrogen sources. While keeping the pH between shake flask and stirred bioreactor comparable, the developed shake flask protocol reproduced the strain behaviour under stirred bioreactor conditions. Compared to a an already existing miniaturized shake flasks protocol, the cellulase concentration was increased 5-fold, reaching about 10 g L-1. Applied to the secondary screening of several clones, the newly developed protocol succeeded in selecting a clone with a high industrial potential.ConclusionsThe understanding of a bioprocess stoichiometry contributed to define a simpler and more effective miniaturization. The suggested strategy can potentially be applied to other fed-batch processes, for the screening of either strain collections or experimental conditions.

Dynamic model of a nitrifying fixed bed column: Simulation of the biomass distribution of Nitrosomonas and Nitrobacter and of transient behaviour of the column

Abstract A dynamic model for a fixed bed nitrifying column with recirculation of the liquid and gas phases was developed. Liquid RTD experiments demonstrated that the liquid phase was perfectly mixed inside the column. Hete- rogeneity of biomass distribution on the solid phase (beads) was represented by an N-tanks in series model, and a back-mixing term was set to account for the well-mixed liquid phase throughout the column. In autotrophic conditions, competition for oxygen is the cause of the spatial segregation of the two species. Nitrosomonas is concentrated on beads at the bottom of the bed whereas Nitrobacter is more widely distributed. This is consistent with biomass distribution results reported by Cox et al. [17] in a nitrifying fixed bed column. Nitrification takes place at the bottom of the column, always in oxygen gas-liquid mass transfer limiting conditions. Nevertheless, considering the whole process, nitrification is complete (>98% of NH3 oxidised) and there is no oxygen limitation (the outlet dissolved oxygen concentration is not limiting). The dynamic behaviour of the column, in conditions set up to avoid biofilm diffusion limitation, was simulated for different NH3-load variations and oxygen shutdowns. The simulated behaviour of the column can be compared to results reported by Bazin et al. [16]. This confirms that the output transient nitrite peaks are higher when changes in the process conditions produce a rearrangement of biomass distribution in the fixed bed.

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.

MELiSSA Higher Plants Compartment Modeling using EcosimPro

This paper introduces two modeling approaches in consideration for the MELiSSA Higher Plants Compartment. This includes an empirical light response curve modeling approach and the Modified Energy Cascade (MEC) model. The MEC model was translated into EcosimPro and evaluated for its performance under a range of environment conditions. The model demonstrated an adequate response to changes on the environmental conditions (temperature, CO 2 concentration and light flux) predicting the gas exchange (O 2 production, CO 2 consumptions, and water vapor transpiration).

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.