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AIR2-CT93-0825
Production of 1,3-propandiol From Glycerol Surpluses. Yield Optimisation By Technological Development and By Genetic Strain Improvement |
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Proposal No: | AIR2-CT93-0825 |
| Date Prepared: | September 1999, April 1998 | |
| Source: | Final report summary 1997 Periodic Progress Report - Year 3 |
Summary
Fermentation of glycerol to 1,3-propanediol (1,3-PD) by Clostridium butyricum can be improved by general optimisation of the culture conditions and by engineering methods increasing the process performance as well as by influencing the metabolic pathways toward higher propanediol yields.
The medium for Clostridium butyricum DSM 5431 was optimised mainly in chemostat culture. It turned out that Clostridium can be grown, up to a dilution rate of 0.4 h-1 without significant decrease in cell density, while in the medium used before the culture almost washed out at this dilution rate. The medium was further improved by reducing the original concentration of ammonium chloride from 5.3 g l-1 to 2 g l-1. The cells were obviously sensitive to ammonia. In order to be able to omit the yeast extract the vitamin requirement was tested. It was shown that only biotin is necessary for growth and that some stimulation was obtained with pantothenate.
These results enabled a defined medium to be established. Using this a productivity was reached which almost approached that of the formerly used medium with one gram per litre yeast extract. Interestingly, the base used for pH control was significant for growth: KOH gave 15 % higher cell densities than NaOH and 50 % higher ones than ammonium hydroxide, the failure of which, as a titrant, was probably due to an increase in the ammonia content.
As the important electron carriers NAD+ and ferredoxin, contain phosphate and iron respectively, continuous cultures under limitation of these nutrients were set up. Limitation by phosphate as well as by iron resulted in an increase of 1,3-propanediol and a decrease of hydrogen production compared to the product limited standard culture. Methyl viologen as an artificial electron carrier had an effect on the product composition similar to that of phosphate or iron limitation: 1.3-propanediol production was increased, and hydrogen production was reduced concomitant with a decrease in acetate formation. In all these cultures growth was considerably affected, and it appears doubtful if these techniques can find application for the improvement of the process, but the results show that the electron flow can be influenced in favour of 1,3-propanediol and that other, preferably genetical methods offer good possibilities.
Cell recycling culture is another rational approach to optimise microbial conversions and hence of cell recycling was applied to the fermentation of glycerol to produce 1,3-propanediol with better efficiency. Crossflow microfiltration with hollow fibre modules was used to retain the cells in the reactor. Optimum operation conditions were found by variation of the medium dilution rate at a fixed retention ratio. At a retention ratio (medium inflow/culture outflow) of 5 the best productivity was achieved at a medium dilution rate of 0.7 h-1 and amounted to 16.6 g l-1 h-1 at a glycerol medium concentration of 56 g l-1. The maximum 1,3-propanediol concentration in the cell-free filtrate was 27 g l-1. These were the highest production values ever reported for continuous fermentation of glycerol. Compared to an unrecycled continuous culture the productivity was increased fourfold. Furthermore, it was found that deterioration of cell activity which usually occurs in cell recycling cultures can almost be avoided if the culture dilution rate approached that of the unrecycled culture.
Glycerol conversion is substantially increased, if glycerol in a concentration of up to 10 g l-1 is retained in the reactor, i.e. the growth conditions are determined by a certain product inhibition.
The presence of high levels of substrate and the accumulation of products in a fermentation broth is known to inhibit the growth of many producer organisms and product formation. Such is the case for Clostridium butyricum for which growth is totally inhibited at concentrations of 788 mM 1,3-propanediol (1,3-PD) and 1,053 mM glycerol. In addition, acetic and butyric acids that are produced are highly toxic at a concentration as low as 70 mM.
Many bacteria have developed mechanisms that allow them to tolerate exposure to multiple stresses such as starvation, pH, osmotic pressure, temperature and redox potential. Using osmotic pressure and pH in the selection procedure, mutants that were more product and substrate tolerant than the parent strain were isolated. Exposure to N-methyl-N-nitro-N-nitrosoguanidine followed by selection of mutants by growing in liquid medium supplemented with toxic levels of 1,3-PD (526 mM) or glycerol (2172 mM), then plating on agar media containing glucose plus an equimolar mixture of bromide and bromate enabled selection of mutants. Better results were obtained with 1,3-PD as selection procedure than with glycerol and 14 mutants with similar properties were obtained. Among them, three representative mutants named 2/2, 2/6 and 5/6 were retained. Isolated mutants were more resistant to glycerol than the wild type: at 1630 mM of glycerol in the medium the percentage of growth inhibition in the wild type was 100 % whereas it was between 38 and 70 % for the mutants. Mutants were also more resistant to 1,3-PD than the wild type: at 395 mM 1,3-PD the percentage of growth inhibition in the wild type was 73 % whereas it was from 10 to 20 % for the mutants.
The major characteristic of these mutants was their ability to produce more biomass. The maximum cell yield for the parent strain was 1.32 g.1-1 compared to 2.9 g.1-1 for the mutant 2/2, 2.0 for the mutant 2/6 and 1.9 for the mutant 5/6. The generation time of the wild type changed from 2.06 hours to an average of 1.7 hours for the mutants. Glycerol and 1,3-PD resistant mutants were selected using the proton suicide method. The acetic and butyric acids produced by the bacteria react with the sodium bromate and bromide salts and release toxic bromine which is lethal for the cells. As expected, the survivors produce less acids than the wild strain.
Product formation for the wild type and the mutants was studied in the culture medium with 60 g l-1 of glycerol. Complete glycerol consumption was observed for the parent strain and the mutants. The total acid formation (acetic and butyric acids) was higher for the wild strain (186 mM) than for the mutants (118-122 mM), the acetate/butyrate ratio decreased from 5 for the parent strain to 0.6-1.0 for the mutants. The conversion of glycerol to 1,3-PD (mol 1,3-PD/mol glycerol) was almost the same: 0.61 for the parent strain and 0.63 for the mutants.
Enzymatic analysis of key metabolic activities were undertaken for glycerol batch cultures (60 g l-1) of C. butyricum and mutants to understand more clearly the nature of the change in the mutant enzymes. Glycerol and 1,3-PD dehydrogenase activities were stable at different phases of growth (exponential phase and end of the growth) in both wild type and mutants. Higher butyrate kinase activities in cell-free extracts of the mutants were in accordance with a decrease in acetate and an increase in butyrate production. The hydrogenase activity increased parallel to growth for the wild type, whereas this activity was stable for the mutants and was markedly decreased by a factor of 5 to 20, which explains the better NADH recovery for the mutants. The NADH ferredoxin reductase activities were significantly lower and the ferredoxin-NAD+ reductase activities were higher for the mutants than the wild type allowing the transfer of the reducing equivalents from the reduced ferredoxin produced by the ferredoxin oxidoreductase to the NADH; transfer facilitated by the low level of the hydrogenase.
The mutant 2/2 was tested in a special fed-batch system which coupled acidification by the excreted acids to the nutrients supply, thus maintaining a low, but consistent glycerol excess in the culture. At the end of fermentation 44 % more glycerol was consumed and 50 percent more 1,3-PD was produced by the mutants than by the wild type. The mutant 2/2 produced about 71 g. l-1of 1,3-PD, the highest concentration ever reported in the literature.
The conversion of glycerol to 1,3-PD (moles of 1,3-PD/moles of glycerol) was slightly better (0.65 for the wild type and 0.69 for the mutants), the excess reducing equivalent being used in butyrate biosynthesis. This demonstrates that the ferredoxin NAD reductase together with acetate-butyrate formation plays a major role in the regulation of the internal redox balance.
Attempts to isolate butyrate negative mutants failed, indicating that C. butyricum cannot be simultaneously hydrogenase negative and butyrate negative. These facts could mean that glycerol catabolism by C. butyricum implicates a rigid first branch point which divides the carbon flux into 1,3-PD and pyruvate biosynthesis and a flexible branch point implicated in acetate and butyrate formation, essential for the regulation of the internal redox balance.
This concept was confirmed by the study devoted to mutants of C. butyricum resistant to allylalcohol (AA). AA could be used to select mutants affected in the path leading to 1,3-PD production. 15 AA mutants were isolated. All mutants produced the same quantities of 1,3-PD demonstrating once more the constancy of the carbon flow at the level of the first branch point and produced more acetate than butyrate. Unlike hydrogenase negative mutants, AA mutants produced more hydrogen than carbon dioxide showing that, in addition to the acetate-butyrate formation, NADH-ferredoxin reductase activity with hydrogenase contributes to the regulation of the electron flow.
The selection of mutants of C. butyricum by allyl alcohol has been used as a tool to isolate mutants that have lost the 1,3-PD dehydrogenase activity. In fact the mutants exhibited almost the same propanediol dehydrogenase activity but changes in the kinetic properties of the enzyme are consistent with mutagenic events leading to an increase in aldehyde reduction and to a decrease in alcohol oxidation and so explain AA-resistance. This demonstrates that glycerol catabolism requires a high 1,3-PD dehydrogenase activity to avoid intracellular accumulation of 3- hydroxypropionaldehyde (3-HPA), a very toxic compound.
The differences in acid production between wild-type C. butyricum E-5 and mutant (the mutant forms 17 % more reducing equivalents) are explained by the hydrogen formation data. Whereas the wild type evolved only trace amounts of molecular hydrogen, the mutant produced more hydrogen than carbon dioxide, 10 % more on average and 80 % more in the most active phase. This is reflected in an unusually low propanediol yield of the mutant. The comparison between the two wild type strains C. butyricum DSM-5431 and C. butyricum E-5 confirmed the higher product tolerance of strain E5 indicated by a prolonged fermentation time. Similar to mutants DSM 5431, E5 is distinguished by low hydrogen production, which is however of little benefit to the final propanediol yield.
The mutants obtained from strain F-5 are of high scientific interest as they demonstrate that an increase of acid production, and therefore production of reducing equivalents, is possible, although in this strain the reducing equivalents are channelled to hydrogen and not to 1,3-propanediol production.
The enzymes glycerol dehydrogenase, diol dehydratase and 1,3-PD dehydrogenase, that constitute the branch point partitioning the carbon flux between 1,3-PD formation and pyruvate were studied in chemostat culture under glycerol limitation. Increasing levels of these enzyme activities with increasing dilution rates, i.e. the carbon flux, expressed a strict regulation and account for the constant proportion of glycerol conversion into 1,3-PD.
High levels of NADH were found in wild-type strain and in mutants. Oxidation of NADH by the 1,3-PD dehydrogenase was linked to the production of 3-HPA by glycerol dehydratase. The fact that high intracellular concentrations of NADH were found means that diol dehydratase activity is the rate-limiting step in 1,3-PD formation, avoiding the accumulation of toxic 1,3-HPA. When propionaldehyde or DL-glyceraldehyde, both substrates of the 1,3-propanediol dehydrogenase were fed into the fermentor with glycerol, this resulted in an increase in (i) glycerol utilisation, (ii) biomass formation and (iii) product biosynthesis.
Aldehyde addition had no significant effect on the repartition of the carbon flow since the glycerol conversion into 1,3-PD was almost the same. The aldehydes decreased the NADP/NAD+ ratios from 4.4 to 2.3 as well as decreasing the pool of NAD+ and NADH; proving that the aldehyde is the factor limiting the 1,3-propenediol dehydrogenase activity. Glycerol dehydratase and 1,3-propanediol dehydrogenase are the two enzymes implicated in the 3-PD biosynthesis and the cells have selected a limited dehydratase activity, an excess of 1,3-propanediol dehydrogenase activity and an excess of NADH to avoid intracellular accumulation of 3 HPA.
Conclusions These results indicate that improvement of the strains by metabolic engineering seems difficult since the enzyme which must be amplified catalyses the formation of a toxic compound. It is clear that these cells have selected a set of elaborate regulations precisely to avoid an excess of 3-HPA formation.
Introduction
In the first two years of the project the work concentrated on the
following aspects:
During the final period work on continuous culture systems were to be carried out in order to increase the productivity of 1,3-propanediol (task 3). For technical reasons, the original plan to use cascade fermentation (task 3a) was replaced by a nutrient controlled fed-batch system. Thus for the final task ( 3b) of the project was the development of a cell recycling system for the 1,3-propanediol production by clostridia. Using various cell recycling methods the rates of product formation rate can usually be increased several times compared to simple continuous culture. In fermentations in which inhibitory products are formed a reduction in inhibition may be expected, as the products are continuously withdrawn from the culture. Nevertheless the extent of product inhibition still determines the success of the fermentation. It is also well known that cell recycling presents a number of technical problems that have to be solved for each culture in a specific way.
Methods
From the various alternatives available, crossflow
microfiltration using hollow fibre modules were selected since such systems had
produced good results for a number of other bacterial fermentations. In order to
find an optimum productivity at the highest possible product concentration
various steady state conditions were investigated under normal and under cell
recycling conditions at varying medium flow rates. Instead of varying medium
dilution rate and cell dilution rate independently two fixed retention ratios
were used. It was expected that, due to product inhibition, the product
concentration will decrease at a considerably lower dilution rate than that at
which the culture is limited only by the maximum growth rate of the cells. The
productivity also depends on the glycerol concentration in the medium, the
effect of which was also investigated.
The enzymes implicated in glycerol catabolism were repressed by inclusion of glucose in the growth medium during the start of the fermentation, there after the level of these enzymes were similar to those found in extracts of cells grown on glycerol. With glucose-glycerol mixtures there was a sharp increase in the conversion of glycerol into 1,3-propanediol. On glycerol alone 57 % of the glycerol was diverted through the 1,3-PD pathway, whereas the theoretical maximum propanediol yield was 72 %. On a mixture of glycerol and glucose, glycerol was used chiefly for the 1,3-PD biosynthesis since 92-93% of the glycerol was converted through the 1,3-PD pathway which explained the high value of glycerol conversion i.e. 0.92 - 0.93 mole of 1,3-PD per mole of glycerol used.
Discussion
From a metabolic point of view these results can be
explained by the fact that the apparent Kms for the glycerol dehydrogenase (GDH)
were markedly higher than those of the glyceraldehyde 3-P dehydrogenase and the
fact that the GDH was more inhibited by NADH than the GAPDH. The kinetic
properties are consistent with a weak GDH activity and hence a shift of the
glycerol carbon flow towards 1,3-PD formation when glucose and glycerol are used
simultaneously. When propionaldehyde or DL-glyceraldehyde, both substrates of
the 1,3-propanediol dehydrogenase were fed into the fermenter with glycerol,
this resulted in an increase in glycerol utilisation, biomass formation and
product biosynthesis. Aldehyde addition had no significant effect on the
repartition of the carbon flow since the glycerol conversion into 1,3-PD was
almost the same (from 0.58 to 0.62.). The aldehydes decreased the NADH/NAD+
ratios from 4.4 to 2.3 as well as the pool of NAD+ and NADH; proving that the
aldehyde level is the factor limiting 1,3 propenadiol dehydrogenase activity.
Glycerol dehydratase and 1,3-propanediol dehydrogenase are the two enzymes implicated in 1 ,3-PD biosynthesis and the cells have evolved a limited dehydratase activity, an excess of 1,3-propanediol dehydrogenase activity and an excess of NADH to avoid intracellular accumulation of 3 HPA a very toxic compound. These results show that 1,3-propanediol formation by C. butyricum can be optimised by metabolic engineering if the gene that encodes for the glycerol dehydratase is amplified. Nevertheless the level of the dehydratase activity must be properly controlled, otherwise toxic levels of 3-HPA could accumulate.
Cell recycling culture is another approach to optimise microbial conversions and this method of cell recycling was applied to the fermentation of glycerol to produce 1,3-propanediol with greater efficiency. Crossflow microfiltration with hollow fibre modules was used to retain the cells in the reactor. Optimum operation conditions were found by variation of the medium dilution rate at a fixed retention ratio. At a retention ratio (medium inflow/culture outflow) of 5 the best productivity was achieved at a medium dilution rate of 0.7 per h and amounted to 16.6 g/l.h at a glycerol medium concentration of 56 g/l. The maximum 1,3-propanediol concentration in the cell-free filtrate was 27 g/l. These were the highest production values ever reported for continuous fermentations of glycerol. Compared to an un-recycled continuous culture, productivity was increased fourfold. Furthermore it was found that deterioration of cell activity which usually occurs in cell recycle cultures can be almost completely avoided by adjusting the culture dilution rate, since growth conditions are determined by product inhibition.
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