Microbial production of organic acids: the expanding markets
Microbial production of organic acids is a promising approach for obtaining building-block chemicals from renewable carbon sources. Although some acids have been produced for some time and in-depth knowledge of these microbial production processes has been gained, further microbial production processes seem to be feasible, but large-scale production has not yet been possible. Citric, lactic and succinic acid production exemplify three processes in different stages of industrial development. Although the questions being addressed by current research on these processes are diverging, a comparison is helpful for understanding microbial organic acid production in general. In this article, through analysis of the current advances in production of these acids, we present guidelines for future developments in this fast-moving field.
Microbial organic acid production: three acids as examples
From Italian lemons to filamentous fungi: citric acid
The oldest microbial process for production of a highvolume, low cost organic acid is the production of citric acid by the filamentous fungus Aspergillus niger. Currently the yearly production of citric acid is approximately 1.6 million tons (t) . Unlike most of the other bio-derived acids that are considered industrial products, citric acid was produced industrially before the development of a microbial process. The industrial production relied on extraction from Italian lemons until it was discovered that Aspergilli accumulate this acid in high amounts under certain conditions. The crucial parameters resulting in efficient production of citric acid by A. niger have been determined empirically and include high substrate concentration, low and finite content of nitrogen and certain tracemetals, thorough maintenance of high dissolved oxygen, and lowpH. The exact definition of these parameters enabled the development of highly efficient biotechnological processes. However, many of the biochemical and physiological mechanisms underlying the process remain unknown. These mechanisms are currently undergoing investigation to enable improvement of the citric acid production process, for which significant improvement is no longer possible through traditional means, such as mutagenesis or cultivation optimization. In addition to the well-established filamentous fungal species, the yeast Yarrowia lipolytica, has been developed as a microbial cell factory for citric acid. The starting point for this line of research was to gain access to n-paraffins and fatty acids (as animal fats) that are not converted by A. niger as carbon sources. However, Y. lipolytica also proved efficient in the production of citric acid from other carbon sources, such as glucose and sucrose. Citric acid concentrations of 140 g/L are now easily reached, and Y. lipolytica is probablyusedonanindustrial scale, although few details are known of actual production methods.
From yoghurt to yoghurt containers: lactic acid
Lactic acid and its production by lactic acid bacteria have a long history in the food industry and microbial processes for lactic acid production were established early in the past century. However, the large-scale commercial production of the purified acid by microorganisms is relatively new. The production of the biodegradable plastic polylactide (used, for instance, in food containers) led to increased interest in optically pure lactic acid. This accounts for the recent shift from chemical to microbial production processes. Approximately 150 000 tons of lactic acid were produced in 2002, 90% of which was by fermentation with lactic acid bacteria. Lactic acid bacteria have complex nutrient requirements and they ferment sugars via different pathways, resulting in homo-, hetero-, or mixed acid fermentation. However, it is not only bacteria that accumulate lactate. The filamentous fungus Rhizopus oryzae is another natural producer that has the advantage of growing on mineral medium and carbon sources such as starch or xylose.
Amber of modern times: succinic acid
The market for succinic (amber) acid is currently small and 16 000 tons per year. However, if the price becomes competitive, succinic acid could replace petroleum-derived maleic anhydride, which has a market volume of 213 000 tons per year. An even higher market volume is conceivable for succinic acid as it is a versatile building-block chemical suitable for many uses. Replacing petroleumderived chemicals, and taking into account that succinic acid formation consumes CO2 (theoretically 1 mole CO2 per mole succinic acid produced) the introduction of succinic acid as a commodity building-block has the potential to lead to large reductions in environmental pollution. To date, no industrial process for microbial succinic acid production has been established; however, calculations show that such a process can be competitive provided that some of the issues outlined below can be resolved. The first approach for microbial production of succinic acid was the engineering of the mixed acid fermentation of Escherichia coli. Later it was discovered that several anaerobic rumen bacteria naturally produce large amounts of succinic acid. However, cultivation of such bacteria is dependent on expensive and complex nutrient sources, and by-product formation is a general problem that remains to be solved . Another interesting strategy was the engineering of a recombinant E. coli by a systems biology approach using a comparison to Mannheimia succiniciproducens, a rumen microorganism, isolated from a Korean cow. However, the yield of succinic acid in the resulting strain was significantly lower than that of the natural producers.