André Bories

1,3-propanediol production by fermentation: An interesting way to valorize glycerin from the ester and ethanol industries

Glycerol is the main by-product from the conversion of agricultural crops into non-alimentary products such as ester and bioethanol. Its microbiological transformation to 1,3-propanediol constitutes a recent approach. The bioproduction of 1,3-propanediol by bacterial species Klebsiella pneumoniae, Citrobacter freundii, Enterobacter agglomerans and Clostridium butyricum have been investigated by batch fermentation and compared at low and high glycerol contents. An important metabolic flexibility was observed with the enterobacteria in contrast to C. butyricum. Fermentation of glycerol by the enterobacterial species revealed the occurrence of an inhibitory phenomenon. It was assigned to the accumulation in the fermentation medium of a strongly inhibitory compound, 3-hydroxypropionaldehyde, the only intermediate of the 1,3-propanediol metabolic pathway. This phenomenon was shown to significantly decrease the biological activities and to be dependent on the culture pH conditions. It was not observed with C. butyricum. By comparing the efficiency of the fermentation among the four bacteria, C. butyricum was chosen for an applied study consisting of the bioconversion of glycerol containing industrial wastewaters. With glycerin coming from the ester production and from wine stillage, a high efficiency of conversion to 1,3-propanediol was observed. The 1,3-propanediol produced was purified by liquid/liquid extraction with a recovery yield about 100%.

Propionic acid fermentation from glycerol : comparison with conventional substrates

Abstract Instead of the conventional carbon sources used for propionic acid biosynthesis, the utilization of glycerol is considered here, since the metabolic pathway involved in the conversion of glycerol to propionic acid is redox-neutral and energetic. Three strains, Propionibacterium acidipropionici, Propionibacterium acnes and Clostridium propionicum were tested for their ability to convert glycerol to propionic acid during batch fermentation with initially 20 g/l glycerol. P. acidipropionici showed higher efficiency in terms of fermentation time and conversion yield than did the other strains. The fermentation profile of this bacterium consisted in propionic acid as the major product (0.844 mol/mol), and in minimal by-products: succinic (0.055 mol/mol), acetic (0.023 mol/mol) and formic (0.020 mol/mol) acids and n-propanol (0.036 mol/mol). The overall propionic acid productivity was 0.18 g l−1h−1. A comparative study with glucose and lactic acid as carbon sources showed both less diversity in end-product composition and a 17% and 13% lower propionic acid conversion yield respectively than with glycerol. Increasing the initial glycerol concentration resulted in an enhanced productivity up to 0.36 g l−1h−1 and in a maximal propionic acid concentration of 42 g/l, while a slight decrease of the conversion yield was noticed. Such a propionic acid production rate was similar or higher than the values obtained with lactic acid (0.35 g l−1h−1) or glucose (0.28 g l−1h−1). These results demonstrated that glycerol is a carbon source of interest for propionic acid production.

Nutrient requirements for glycerol conversion to 1,3-propanediol by Clostridium butyricum

A low-nutrient medium (LNM) based on biotin as the sole growth factor was defined to extend nutritional knowledge and to give more precise details about essential nutrients for Clostridium butyricum. Batch fermentations were performed with glycerol or industrial glycerin as a carbon source. It was shown that only 4 μg/l of biotin was sufficient to convert up to 129 g/l of glycerol and 121 g/l of industrial glycerin into a large amount of 1,3-propanediol; up to 67 g/l (0.63 mol/mol glycerol used) and 65 g/l (0.66 mol/mol), respectively. In addition, with the proposed medium, evidence was provided that nitrogen could constitute a limiting factor for glycerol fermentation, especially when the C/N ratio was less than 81:1. Despite its simple composition, the synthetic medium described here gives the same performance for C. butyricum on industrial glycerin as does a rich medium (RM) based on yeast extract.

Glycerol fermentation by a new 1,3-propanediol-producing microorganism:Enterobacter agglomerans

According to their ability to synthesize 1,3-propanediol from glycerol, two species were isolated from the anoxic mud of a distillery waste-water digestor:Clostridium butyricum andEnterobacter agglomerans. The latter, a facultatively anaerobic gram-negative bacterium, is described for the first time as a microorganism producing 1,3-propanediol from glycerol. The products of glycerol conversion byE. agglomerans were identified using nuclear magnetic resonance. A 20-g/l glycerol solution was fermented mainly to 1,3-propanediol (0.51 mol/mol) and acetate (0.18 mol/mol). Ethanol, formate, lactate and succinate were formed as by-products. Gas production was very low; 1,3-propanediol production perfectly balanced the oxido-reduction state of the microorganism. Acetate was the predominant metabolite generating energy for growth. High-glycerol-concentration fermentations (71 g/l and 100 g/l) resulted in an increase of the 1,3-propanediol yield (0.61 mol/mol) at the expense of lactate and ethamol production. Specific rates of glycerol consumption and 1,3-propanediol and acetate production increased whereas the growth rate decreased. The decreased in ATP yield was linearly correlated with the specific rate of 1,3-propanediol production. Incomplete glycerol consumption (about 40 g/l) was systematically observed when high glycerol concentrations were used. The unbalanced oxido-reduction state, the low carbon recovery and the detection of an unknown compound by HPLC observed in these cases indicate the formation of another metabolite, which is possibly an inhibitory factor.

Effects of Acetate and Butyrate During Glycerol Fermentation by Clostridium butyricum

The effects of acetate and butyrate during glycerol fermentation to 1,3-propanediol at pH 7.0 by Clostridium butyricum CNCM 1211 were studied. At pH 7.0, the calculated quantities of undissociated acetic and butyric acids were insufficient to inhibit bacterial growth. The initial addition of acetate or butyrate at concentrations of 2.5 to 15 gL−1 had distinct effects on the metabolism and growth of Clostridium butyricum. Acetate increased the biomass and butyrate production, reducing the lag time and 1,3-propanediol production. In contrast, the addition of butyrate induced an increase in 1,3-propanediol production (yield: 0.75 mol/mol glycerol, versus 0.68 mol/mol in the butyrate-free culture), and reduced the biomass and butyrate production. It was calculated that reduction of butyrate production could provide sufficient NADH to increase 1,3-propanediol production. The effects of acetate and butyrate highlight the metabolic flexibility of Cl. butyricum CNCM 1211 during glycerol fermentation.

Physiology of Gluconobacter oxydans during dihydroxyacetone production from glycerol

Investigations into physiological aspects of glycerol conversion to dihydroxyacetone (DHA) by Gluconobacter oxydans ATCC 621 were made. The activity levels of the enzymes involved in the three catabolic pathways previously known and the effects of specific inhibitors and uncoupling agents on cellular development, DHA synthesis, and cellular respiratory activity were determined. It was established that only two catabolic pathways are involved in glycerol dissimilation by this micro-organism. The only enzyme responsible for DHA production is membrane-bound glycerol dehydrogenase, which employs oxygen as the final acceptor of reduced equivalents without NADH mediation. The ketone is directly released into the culture broth. As the glycolytic and carboxylic acid pathways are absent, the pathway provided by the membrane-bound enzyme is indispensable for the energy requirements of G. oxydans. The cytoplasmic pathway, which begins by phosphorylation of glycerol followed by a dehydrogenation to dihydroxyacetone phosphate, allows growth of the bacterium. At the same time, the substrate transport mode was characterized as facilitated diffusion using radioactive [1(3)-3H]-glycerol. Concerning the DHA inhibition of microbial activity, the enzymatic study of the membrane-bound glycerol dehydrogenase showed the enzymatic origin of this phenomenon: a 50% decrease of the enzyme activity was observed in the presence of 576 mm DHA. The decrease in the rate of penetration of glycerol into cells in the presence of DHA indicates that growth inhibition is essentially due to the high inhibition exerted by the ketone on the substrate transport system.

Glycerol inhibition of growth and dihydroxyacetone production byGluconobacter oxydans

The evidence, kinetic aspects, and modelization of the inhibitory effect of glycerol on dihydroxyacetone (DHA) production byGluconobacter oxydans have been studied. The comparison of the maximal productivities and specific rates evaluated for initial concentrations of 31, 51, 76, 95, and 129 g L−1 of substrate showed that glycerol exerts an inhibitory effect both on growth and DHA production: decrease of the growth-specific rate and of the specific rate of DHA production with increase of the initial glycerol content. The inhibition phenomenon was attributed to an immediate effect of glycerol on the biological activity. It was also established that the presence of glycerol at high concentration induces an increase in the time necessary for the cells to reach their maximal level of specific rates. This result tends to show that glycerol brings into play on the biological system the capacity to reach its optimal range of activity. The main models found in the literature dealing with substrate inhibition phenomena were then tested on experimental data. The exponential model describes at best the glycerol inhibition on growth (μ=0.53e(−S/93.6)) and on DHA production (qP=7e(−S/76.7)). The kinetic study and modelization of the inhibition effect of glycerol on DHA production allows one, therefore, to fill the gap in the fundamental knowledge of this industrial fermentation, to show the maladjustment of the classical fermentation process used (batch), and to reconsider the conception for the optimization of the production (proposition of more adapted process like fed-batch and/or biphasic systems).

Anaerobic pathways of glycerol dissimilation by Enterobacter agglomerans CNCM 1210 : limitations and regulations

Continuous cultures of Enterobacter agglomerans CNCM 1210 were performed under regulated pH conditions (pH 7.0) with glycerol or glucose (20 g l-1) as carbon source. Cultures grown on glucose produced mainly acetate, ethanol and formate. In contrast, 1,3-propanediol (PPD) was the main product with glycerol. The carbon flow distribution at branching metabolic points was investigated. Higher PPD yields with increased dilution rate were correlated with an important increase in the relative ratio of glycerol dehydratase to glycerol dehydrogenase. Determination of intracellular triose-phosphate and fructose 1,6-biphosphate concentrations demonstrated that glyceraldehyde-3-phosphate dehydrogenase is the limiting step in glycerol dissimilation. At the pyruvate branching point, pyruvate dehydrogenase (PDH) activity was systematically detected. The pyruvate flow shifted to PDH is suspected to represent up to 22% of the acetyl-CoA formed. In addition, this enzyme pattern combined with the enhanced in vivo lactate dehydrogenase activity at high growth rates, was correlated with a decrease in the pyruvate formate-lyase activity. A regulation of this latter enzyme by the accumulation of triose-phosphate is suspected.

Kinetic study and optimisation of the production of dihydroxyacetone from glycerol using Gluconobacter oxydans

Abstract Kinetic parameters were determined during dihydroxyacetone (DHA) production by Gluconobacter oxydans in batch culture with different glycerol concentrations (50 and 100 g litre−1). Inhibitory effects were related both to substrate and product. When the initial glycerol concentration was increased from 50 to 100 g litre−1, all the specific rates and productivities were affected and were lower than the values obtained with an initial glycerol concentration of 50 g litre−1. The kinetic parameters were optimal in fed-batch culture; a higher amount of glycerol being converted compared to a batch culture performed with initial substrate concentration of 100 g litre−1. DHA exerted an inhibitory effect on G. oxydans which was more pronounced and more selective than the actin of glycerol. The rate of growth decreased with increasing DHA concentration, and finally ceased at a DHA concentration of 61 g litre−1. Glycerol oxidation into DHA was not affected during this period suggesting a decoupling of growth and production. When the DHA concentration in the culture medium reached 108 g litre−1, then glycerol conversion ceased. Mannitol was a preferred substrate and batch growth on mannitol followed by fed-batch DHA production on glycerol avoided the inhibitory effects due to substrate and product and led to an optimised DHA production process.

Interest of electrodialysis to reduce potassium level in vinasses. Preliminary experiments

Nowadays, treatment of vinasse from the alcohol fermentation is a major cost centre which decides the economic viability of this traditional industry. Increasingly stringent environment norms in France have put enormous pressure on manufacturers of alcohol from traditional feedstock like beet molasses. Nowadays, the most industrial treatment is the vinasse valorisation as fertiliser in the field after concentration. Nevertheless, the final solids are limited because of the sulphate potassium crystallisation and precipitation in evaporator tubes, storage tanks and fertiliser sprayers. The paper gives some figures of beet molasses vinasse composition, details the most widely used treatment schemes and presents the first results obtained with an electrodialysis treatment to reduce the potassium concentration in view to prevent the crystallisation and even increase the final dissolved solids of the concentrated vinasse.