Rémy Marchal

Identification and production of a rhamnolipidic biosurfactant by a Pseudomonas species

A glycolipid-producing bacterium, Pseudomonas aeruginosa GL1, was isolated from the soil contaminated with polycyclic aromatic hydrocarbons (PAH) from a manufactured gas plant. The glycolipid produced was characterized in detail by chromatographic procedures as a mixture of four rhamnolipids, consisting of different associations of rhamnose and hydroxy fatty acids: the main component was monorhamnosyl di-3-hydroxydecanoic acid. The rhamnolipid composition presented marked analogies with a defined part of P. aeruginosa outer membrane lipopolysaccharides (lipopolysaccharide band A). Rhamnolipid production was stimulated under conditions of nitrogen limitation. Glycerol yielded higher productions than did hydrophobic carbon sources. Cell hydrophobicity decreased during growth on glycerol and on n-hexadecane whereas glycolipid production increased. P. aeruginosa GL1 was found to be unable to grow on a variety of 2, 3 and 4 cycle PAH. However, it was shown to persist after at least 12 subcultures in a bacterial population growing on a mixture of pure PAH, suggesting a physiological role for rhamnolipid as a means to enhance PAH availability in a mutualistic PAH-degrading bacterial community.

Kinetics and balance of a fermentation free from product inhibition : sophorose lipid production by Candida bombicola

SummaryThe production of sophorose lipids from ethyl esters of rapeseed oil fatty acids and glucose by Candida bombicola CBS 6009 was studied. A kinetic description of the fermentation is presented, showing in particular two distinct phases of growth and, following nitrogen limitation, subsequent production of sophorose lipids, which were excluded from the aqueous phase. With the fed-batch fermentation technology used, a high production performance (320 g·l−1 sophorose lipids with a weight yield of 65% with respect to the carbon sources) was obtained. Detailed fermentation balances were established and their implications for energy metabolism during glycolipid production are discussed. The variations in the structure of the sophorose lipids during fermentation were also studied. Sophorolipid 1′, 4″-lactone 6′, 6″-diacetate was the major class compound produced (about 50% of the glycolipids). A correlation between the lipidic composition of the products and the nature of the fatty acids of the rapeseed esters was observed.

Biodegradation of gasoline: kinetics, mass balance and fate of individual hydrocarbons

The degradation of gasoline by a microflora from an urban waste water activated sludge was investigated in detail. Degradation kinetics were studied in liquid cultures at 30 °C by determination of overall O2 consumption and CO2 production and by chromatographic analysis of all 83 identifiable compounds. In a first fast phase (2 d) of biodegradation, 74% of gasoline, involving mostly aromatic hydrocarbons, was consumed. A further 20%, involving other hydrocarbons, was consumed in a second slow phase (23 d). Undegraded compounds (6% of gasoline) were essentially some branched alkanes with a quaternary carbon or/and alkyl chains on consecutive carbons but cycloalkanes, alkenes and C10‐ and C11‐alkylated benzenes were degraded. The degradation kinetics of individual hydrocarbons, determined in separate incubations, followed patterns similar to those observed in cultures on gasoline. Carbon balance experiments of gasoline degradation were performed. The carbon of degraded gasoline was mainly (61·7%) mineralized into CO2, the remaining carbon being essentially converted into biomass.

Control of the selectivity of butyric acid production and improvement of fermentation performance with Clostridium tyrobutyricum

SummaryThe parameters that control fermentation performance of butyrate production have been studied with a selected strain ofClostridium tyrobutyricum. Fed-batch supply of glucose increased productivity for butyrate. The ratio of butyrate to total acids was strongly influenced by the growth rate of the bacteria, acetate being produced along with butyrate at higher growth rates. In glucose-limited, fed-batch cultures, initially produced acetate was re-utilized, resulting in exclusive production of butyrate. In cultures with non-limiting glucose feeding, the butyrate concentration reached 42.5 g·1−1 with a selectivity of 0.90, a productivity of 0.82 g·−1 per hour and a yield of 0.36 g·g−1 The effects of the mode of supply of glucose on the production of butyrate and acetate are discussed in relation with the energy requirements for cell growth.

A Mycobacterium Strain with Extended Capacities for Degradation of Gasoline Hydrocarbons

ABSTRACT A bacterial strain (strain IFP 2173) was selected from a gasoline-polluted aquifer on the basis of its capacity to use 2,2,4-trimethylpentane (isooctane) as a sole carbon and energy source. This isolate, the first isolate with this capacity to be characterized, was identified by 16S ribosomal DNA analysis, and 100% sequence identity with a reference strain of Mycobacterium austroafricanum was found. Mycobacterium sp. strain IFP 2173 used an unusually wide spectrum of hydrocarbons as growth substrates, including n-alkanes and multimethyl-substituted isoalkanes with chains ranging from 5 to 16 carbon atoms long, as well as substituted monoaromatic hydrocarbons. It also attacked ethers, such as methyl t-butyl ether. During growth on gasoline, it degraded 86% of the substrate. Our results indicated that strain IFP 2173 was capable of degrading 3-methyl groups, possibly by a carboxylation and deacetylation mechanism. Evidence that it attacked the quaternary carbon atom structure by an as-yet-undefined mechanism during growth on 2,2,4-trimethylpentane and 2,2-dimethylpentane was also obtained.

Sophorose lipid fermentation with differentiated substrate supply for growth and production phases

Abstract Sophorose lipid production by Candida bombicola is a two-step process where sophorose lipids are mainly produced after a first stage of growth, ending because of nitrogen limitation. The influence of the following parameters was individually studied for both the stages of growth and of product formation with respect to final sophorose lipid production performance: pH, temperature and carbon source. Glucose and rapeseed ethyl esters were supplied individually or as a dual carbon source. The lipidic substrate was added by continuous feeding. It was found that supplying both carbon sources during the production step was crucial for obtaining a high production performance ranging from 250 g l−1 to 300 g l−1 or more. Controlling the feeding of rapeseed ethyl esters to avoid inhibition by fatty acids was essential for a successful scale-up of the fermentation on the industrial scale. The conditions of substrate feeding markedly affected the composition of the mixture of sophorose lipids produced, namely the extent of acetylation of the sophorose moieties and distribution of the acidic and lactonic forms. The results suggest that the physiological role of sophorose lipid production is related to the regulation of energy metabolism.

Sophorose lipid production from lipidic precursors: Predictive evaluation of industrial substrates

SummarySophorose lipids stand out as biosurfactants with a wide potential for industrial application and which can be produced in good yield from glucose and a lipidic cosubstrate.Candida bombicola CBS 6009 (ATCC 22214) was used in the present study. The influence of the lipidic cosubstrate on various aspects of production performance of these glycolipids (final concentration, yield) and on product composition (in particular, the structure of the hydroxy fatty acid vegetable and animal oils, markedly influenced product composition. In terms of production performance, the best substrates were oils or esters rich in C18:0 and C18:1 fatty acids. Optimal overall performance was obtained with esters (340 g L−1 sophorose lipids with rapeseed esters). Conclusions drawn from the results allow predictive evaluation of lipidic industrial substrates.

Large-scale enzymatic hydrolysis of agricultural lignocellulosic biomass. Part 1: Pretreatment procedures

Abstract A process for conversion of lignocellulosic biomass into acetone-butanol (ABE) involving steam explosion pretreatment of the raw material, cellulase production, enzymatic hydrolysis and acetone-butanol fermentation of hydrolysates has been developed. Procedures and scale-up of the steam-explosion pretreatment of corncobs are described. The influence of experimental parameters, time, temperature and addition of acids, on both the performance of enzymatic hydrolysis and the fermentability into ABE of the hydrolysates obtained were first studied in a batch pilot reactor. The same criteria of performance were applied in large scale development work which was completed using a continuous industrial plant (Stake II machine with a capacity of 2–4 t per h) located in the biomass conversion facilities at Soustons, France. Under optimal conditions, which were strongly dependent on the technology used, nearly quantitative hydrolysis yields could be obtained with moderate amounts of appropriate cellulase preparations.

Industrial optimization of acetone-butanol fermentation: a study of the utilization of Jerusalem artichokes

SummaryAcetone-butanol fermentation of the Jerusalem artichoke has been studied as a case for systematic investigation of the industrial optimization of both strain selection and fermentation operation. Hydrolysis of the inulinic oligofructans of the substrate was found necessary for optimal performance but could be achieved with a selected strain using a moderate amount of inulinase added at the beginning of the fermentation. Apart from ammonia, no nutritional supplementation of the medium was found necessary. The marked influence of pH in the fermentation performance prompted a detailed search for a method of controlling pH during fermentation. With an optimized procedure, solvent production of 23–24 g/l were obtained in 36 h. Detailed fermentation balances are presented. An industrial process for ABE production from Jerusalem artichoke or sugar beet has been defined and tested in the pilot plant.

Identification and determination of individual sophorolipids in fermentation products by gradient elution high-performance liquid chromatography with evaporative light-scattering detection.

High-performance liquid chromatography (HPLC) was used for the characterization of sophorolipids, one of the most important types of glycolipid biosurfactants. By using gradient elution with a water-acetonitrile mixture on a reversed-phase (C18) column and evaporative light-scattering detection, resolution of all the important individual sophorolipids present in fermentation products was achieved. In addition to HPLC, a combination of techniques involving selective production by fermentation of sophorolipids, chemical conversions of the products, separation methods and, for identification of lipidic chains of sophorolipids, gas chromatography and mass spectrometry was used. This led to the identification of almost all significant compounds observed in HPLC, including several previously unreported sophorolipids. As a result, a rapid method is now available for investigations of the influence of fermentation conditions on the nature and quantitative distribution of the sophorolipid products obtained.