It is still an open question if the enterobacteria of the genera Klebsiella and Citrobacter or the Clostridia are more suitable for a 1,3-PD production process. From the viewpoint of an interested company there will probably be a preference for the clostridia as both enterobacterial species are classified as opportunistic pathogens and thus would require costly safety precautions. On the other hand, as shown in the preceding section the strains of Clostridium butyricum which are presently in use cannot entirely compete with strains like Klebsiella pneumoniae DSM 2026 at least if productivity is concerned (38). Several efforts have been made in the last five years to meet this shortcoming. The medium has been improved, better culture techniques have been elaborated, new strains have been isolated from nature or selected from culture collections, and mutants have been obtained which are considerably increased in product tolerance and yield.

Medium and Culture Conditions. For estimations of the performance of Klebsiella and Clostridium cultures virtually different media had been in use. When the medium used for Klebsiella (14) was applied to Clostridium butyricum DSM 5431 indeed the steady state product concentration in continuous cultures could be increased. Higher iron con­tent and the presence of citrate as a complexing agent were found to be the main cause of this stimulation (24). The yeast extract formerly used in a concentration of 1 g/1 (17) could be replaced by biotin with only a slight loss in productivity.

Continuous culture is a good tool for anaerobic glycerol fermentation particularly when a second stage is used to increase the final product concentration (8). However, for maximum propanediol content and simple operation batch cultures appear to be mo­re advantageous. Giinzel et al. (17) described a fedbatch culture that consumed about 110 g/1 of glycerol supplied in three successive additions over a period of 24 h which resulted in a 1,3-propanediol concentration of 56 g/1. Recently this process was automa­ted using the pH decrease as a signal for glycerol addition. The culture was kept at a slight glycerol excess so that substrate limiting intervals were avoided; the residual gly­cerol was used up towards the end of the fermentation after turning off the glycerol supply. The results were about the same as in discontinuous feeding (24a). A similar system was described by Saint-Amans et al. (27) for C. butyricum VPI 3266 using C02 production for control of glycerol supply. Probably due to the properties of the particu­lar strain (see below) the final product concentration was higher (65 g/1), but the fer­mentation time was about three times as long.

Procedures to increase the productivity by immobilization or cell recycling have been rarely elaborated for the glycerol fermentation. Pflugmacher and Gottschalk (23) described a fixed bed loop reactor culture of Citrobacter freundii using polyurethane foam as carrier substance. In comparison to a stirred tank reactor culture (8) the propa­nediol productivity was more than doubled, but the propanediol concentration could not be increased beyond 19 g/1. Similar results were obtained with Clostridium butyri — cum using a crossflow filtration module. Although it was possible to increase the pro­ductivity up to 3 times of the conventional continuous culture, the steady state propane­diol concentration was not higher than 14 g/1 (Reimann and Biebl, unpublished results).

Use of Cosubstrates. As pointed out above a 1,3-propanediol yield of 72 mol per 100 mol of glycerol cannot be surpassed. If however another fermentable electron donor substrate could be applied in addition to glycerol, a 100 % product yield is conceivable. In the patent literature procedures are described in which glucose is the cosubstrate and enterobacteria the converting organisms. It is proposed to grow the cultures initially with glycerol alone to induce the 1,3-propanediol forming enzymes. Glucose is added either directly to the growing culture (32) or used in a mixture with glycerol in a resting-cell culture without an ammonium source (16). 91 to 100 % of the glycerol were converted to 1,3-propanediol by this technique.

If glucose is considered as cosubstrate it has to be kept in mind that hexoses are less reduced substrates than glycerol, so that twice as much glucose than glycerol is needed on a weight basis to provide the same amount of reducing equivalents. This im­plies that use of glucose in 1,3-propanediol production would only be useful if glucose is available at a considerably lower price than glycerol, which appears to be presently the case, especially in the USA.

Another prerequisite for glucose addition should be that it is converted mainly to acetate in the presence of glycerol and not to ethanol or butyrate. This has been verified for C. butyricum (6). However, there are other physiological barriers in simultaneous metabolization of glycerol and glucose. As reported by Biebl and Marten (6) glucose is fermented much slower than glycerol by glycerol-fermenting Clostridia so that a com­plete conversion of the glycerol cannot be obtained. A fermentable substance other than glucose which is a cheaply available chemical bulk product, ethylenglycol, proved to be unsuitable as a cosubstrate. In the presence of glycerol this diol did not release reducing equivalents in the course of acetate formation as expected, but utilized hydrogen equi­valents to yield ethanol as product, while glycerol was oxidized to acetate.

New Strains of Clostridium butyricum. Whereas in the case of Klebsiella pneumoniae the type strain of the species is still unsurpassed in glycerol fermentation, in the case of Clostridium butyricum new screening efforts have led to strains that are markedly im­proved in product tolerance (22). In fed batch culture the best strain (E5) produced up to 66 g/1 of propanediol from 122 g/1 of glycerol compared to 55 g/1 from 108 g/1 of glyce­rol with strain DSM 5431 (5,17). The new strain was distinguished by very low hydro­gen evolution (24a). However, its maximum growth rate and productivity were distinct­ly lower. The strain used by Saint-Amans et al. (26), C. butyricum VPI 3266, is proba­bly closely related.

Genetic Approaches to Strain Improvement in C. butyricum have been undertaken only very recently (22). NTG mutations of strain DSM 5431 were selected in the pre­sence of high propanediol concentrations and further on bromide-bromate glucose me­dium to obtain mutants that sustained substantially higher product concentrations and were strongly reduced in hydrogen evolution (Reimann and Biebl, unpublished data). In fedbatch culture the best of them was able to convert up to 130 g of glycerol to about 70 g of 1,3-propanediol. Thus it resembles the new isolates obtained by the same group as well as the VPI strain used by Saint-Amans et al. (27,28). If these strains will be sub­jected to genetic improvement further increase in product concentration can be expec­ted.

Attempts to generate a strain from the product tolerant, hydrogen reduced mutants that was defect or affected in butyrate formation failed with several methods. In contrast it was possible, to obtain butyrate reduced mutants from the isolate E5 by selection on allylalcohol (22). These mutants were not much changed in propanediol production but exhibited a hydrogen production that was near the physiological maximum whereas the parent strain was very low in hydrogen evolution. This result in addition to fermenta­tion data and experiments that showed stimulation of 1,3-propanediol production in the presence of an aldehyde prompted the authors to assume that glycerol dehydration the product of which is the toxic 3-hydroxypropionaldehyde is the rate limiting step. Con­sequently, a certain amount of reducing equivalents has to be disposed via butyrate and/or hydrogen, as the 1,3-propanediol pathway can be only varied within a narrow range. A possibility to claim all reducing equivalents produced in glycolysis for 1,3- propanediol formation would be to overexpress the glycerol dehydratase along with the

1.3- propandiol dehydrogenase using recombinant DNA techniques. Such experiments are presently in progress.

Genetic Improvement with Enterobacterial Strains. From Citrobacter and Klebsiella strains over-producing mutants have not been isolated. On the other hand basic genetic research on the key enzymes of the process is more advanced than in Clostridia. In both species the genes of the dha regulon which encode for the glycerol dehydrogenase, the dihydroxyacetone kinase, the gycerol dehydratase and the 1,3-propanediol dehydroge­nase have been cloned and expressed in E. coli (10,11,30,31). A new construction of the dha regulon has been achieved by Cameron (personal communication). The amounts of

1.3- propanediol wich have been reached in the recombinant E. coli strains do not yet approach that of the original strains (i. e. 9 g/1 for strains with Citrobacter and 6 g/1 for strains with the Klebsiella genes), but in the long term highly productive recombinant Klebsiella strains can be expected as soon as the optimized genes have been reintrodu­ced into the donor strains.