Research Article: A co-utilization strategy to consume glycerol and monosaccharides by Rhizopus strains for fumaric acid production

Date Published: April 30, 2018

Publisher: Springer Berlin Heidelberg

Author(s): Sylwia Kowalczyk, Elwira Komoń-Janczara, Agnieszka Glibowska, Adam Kuzdraliński, Tomasz Czernecki, Zdzisław Targoński.


The ability of Rhizopus oryzae to produce fumaric acid in the presence of glycerol and/or various monosaccharides as carbon sources was examined for seventeen different strains of this fungi. These strains were tested in shake-flask cultures on media containing glycerol and seven different carbohydrates, including glucose, fructose, galactose, mannose, xylose, arabinose, and rhamnose. An interesting and applicationally useful phenomenon was observed. This work presents a new approach to the conventional microbiological method of producing fumaric acid. In the presence of 40 g/l glycerol as the sole carbon source, fumaric acid production reached 0.16–6.1 g/l after 192 h. When monosaccharides were used as a single carbon source, the maximum fumaric acid concentration was much higher; for example, 19.8 g/l was achieved when 40 g/l xylose was used. In the co-fermentation of xylose (40 g/l) and glycerol (20 g/l), post-culture broth contained approx. 28.0 g/l of fumaric acid with a process yield of 0.90 g/g after 168 h. The production of fumaric acid by Rhizopus oryzae was also increased in the dual presence of glycerol and monosaccharides like fructose, galactose, and mannose. However, results obtained on glucose-glycerol-based medium did not follow this trend, showing instead complete utilization of glucose with significant glycerol consumption, but unexpectedly low final amounts of fumaric acid and process yields. Understanding how Rhizopus oryzae utilize various carbon sources may provide alternative avenues of fumaric acid fermentation.

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In industrial biotechnology, feedstock is by far the highest cost factor in the production of chemicals, representing 40–60% of total costs (Demain 2007). Therefore, there is an increasing interest in using food waste products, crude glycerol, and lignocellulosic materials as a feedstock for biotechnological processes. Lignocellulosic biomass is composed of three main compounds: cellulose, hemicelluloses, and lignin. Cellulose is a polymer of glucose, and thereby a potential source of fermentable sugar. Hemicellulose hydrolysates consist mainly of xylose, glucose, mannose, arabinose, galactose, traces of other sugars, uronic acid, and acetyl groups, and they offer an attractive possibility to be used as a substrate in fermentation processes. Biodiesel production generates about 10% glycerol as the main byproduct. In recent years, the price of crude glycerol decreased from about 0.25 USD per pound to 0.05 USD per pound. Worldwide, the source of crude glycerol derived from biodiesel conversion increased from 200,000 tons in 2004 to 1,224,000 tons in 2008 (Yang et al. 2012). According to new market research, the global glycerol market is expected to reach 3 billion USD by 2022 (; Therefore, it is important to find new applications for crude glycerol. It has been reported that glycerol is a good carbon source for many microorganisms, such as fungi and bacteria involved in the production of value-added fuels and chemicals (da Silva et al. 2009; Nicol et al. 2012).

Different carbon sources had a significant impact on fumaric acid production by Rhizopus strains. For most studied strains, glucose was the poorest source of carbon for fumaric acid production compared to fructose and galactose. Moon et al. (2004) examined the effect of different carbon sources on fumaric acid production by Rhizopus sp. The highest concentration of fumaric acid was found in the filtrates after Rhizopus sp. culture in the presence of rice bran with the addition of glucose and fructose, followed by maltose, starch and galactose, and lastly, glycerol and lactose. In contrast, no fumaric acid was found in post-culture filtrates when sucrose was used as the carbon source. Hemicellulose is one of the most readily available renewable resources that can be hydrolyzed to produce a mixture of monosaccharides containing xylose, arabinose, and rhamnose. There have been several reports on xylose utilization by Rhizopus sp. for fumaric acid production (Kautola and Linko 1989; Liu et al. 2015). Kautola and Linko (1989) demonstrated that the highest fumaric acid concentration reached with immobilized cells was 16.4 g/l at 10% initial xylose and a residence time of 10.25 days. In this study, titers obtained on xylose for over half of studied strains were higher, and the culture duration was shorter than previously stated by Kautola and Linko (1989).