Short Communication. Citric Acid Fermentation and the Effects of Temperature

Transcription

1 Acta Biotechnol. 23 (2003) 1, Short Communication Citric Acid Fermentation and the Effects of Temperature SELAHZADEH, R. M., ROEHR *,M. Technische Universität Wien * Corresponding author Institut für Verfahrenstechnik, Umwelttechnik Fax: und Technische Biowissenschaften mroehr@mail.zserv.tuwien.ac.at Getreidemarkt 9/E Wien, Austria Summary The temperature of the fermentation media heavily affects yields as well as rates of citric acid fermentations. At C, more than 40% of the available carbohydrate raw material is wasted, apparently through the respiratory activity of the fungus. At C, these losses are decreased to about 10%. The results thus emphasise the economic importance of adequate temperature control in industrial citric acid fermentation. Introduction It has been demonstrated in recent reports [1, 2, 3] that rapid and high-yielding citric acid fermentations may be attained if the following parameters are optimised at the beginning of the respective fermentation runs: Manganese concentration should be adjusted to values below or close to 1 µg/l; iron content should be adjusted to µg/l; growth of the fungus, a selectant of Aspergillus niger, should be limited to values between 10 and 15 g/l (d.wt.) by adjusting the phosphorus concentration to about 25 mg/l. Provided that these conditions can be realised, productivities well above 1g/l. h (space-time yields above 0.6 g/l. h) and yields approaching about 90% (considering the consumption of sugar for biomass production) can be achieved. As shown, however, in one of the above-mentioned reports [2], a particular reason for decreased yields may be the uncontrolled respiratory consumption of sugar by the fungal biomass leading to appreciable waste of the carbohydrate raw material. In the present communication, experiments studying the effects of varied temperatures on the consumption of the carbohydrate source demonstrating that temperature significantly affects the respiratory activity of the fungus are reported. WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim, /03/ $ /0

2 96 Acta Biotechnol. 23 (2003) 1 Materials and Methods The production strain was a selectant of Aspergillus niger ATCC 11440, selected for its high citric acid yield by repeated cultivation of large numbers of single spore cultures in shake flasks using a standard medium (cf. [1]). The bioreactor was an all-plastic 1-L magnetic culture vessel as sold by the NALGE COMPANY, USA (Cat. No. 2605), using a Variomag Mobil 60 magnetic stirrer. Fermentation medium and analytical procedures were essentially as described in [1]. The fermentation procedure was also described in [1], with the exception that the stirred reactor with high stirring rates ( rpm) required higher amounts of antifoam (e.g. Glanapon DC 180) to be added. Dissolved oxygen concentration was measured polarographically using a common INGOLD electrode. Generally, it was found to be sufficient to maintain an oxygen concentration of the medium above 50% of saturation throughout the experiment by adjusting stirring rate as well as aeration rate accordingly. Temperature was controlled in a water bath using a common thermostat and measured by a multichannel thermometer (Consort T851). The technique described enables fermentations with high productivities and yields as will be shown below. In order to establish stoichiometric balances with regard to the fate of the carbon source during the respective experiments, a special kind of presentation was chosen (cf. [2]): Sugar consumption is quoted as percentage of total sugar input. Citric acid production is quoted as sugar consumption (percentage of total) to produce the respective amount of citric acid according to the stoichiometry of the reaction C 6 H 12 O O 2 = C 6 H 8 O H 2 O Biomass is quoted as the respective sugar consumption (percentage of total), taking into account a molecular weight of fungal biomass as 157 g per mole (including an ash content of 9%) according to an empirical equation for biomass formation (cf. e.g. [4]) 2C 6 H 12 O O 2 + NH 3 = C 6 H 10 O 3 N + 6 CO H 2 O resulting in a yield coefficient of 0.44 g biomass d.wt./g sugar. The value thus obtained is added to that of citric acid to draw the respective curve. The advantage of this kind of quotation is that it is possible to check whether a particular fermentation has resulted in a complete conversion of the carbon source to the desired product(s) or whether there was formation of by-products. Apparently, the most likely by-product is CO 2 as the product of undesirable respiration of the carbon source. Results Fermentations at Temperatures above 30 C Fermentations were performed with initial glucose concentrations of g/l (0.87 M) and g/l (0.88 M), respectively. Temperature was held at 32 C M (118.8 g/l) and 0.63 M (121.5 g/l), respectively, of citric acid were formed within a fermentation time of about 200 hours, corresponding to a space-time yield of about 0.60 g/l. h. Citric acid yields were 75.5 and 76.3 g/100 g initial sugar, respectively. Considering the amount of the carbohydrate source being used for biomass formation as mentioned above (0.14 and 0.15 M, respectively), 87 or 88%, respectively, of the carbohydrate source were utilised, i.e. 13 or 12%, respectively, were wasted. The course of one of the fermentations is shown in Fig. 1. The situation changes appreciably if the temperature is adjusted to values around 26 C, as shown below.

3 SELAHZADEH, R.M.,ROEHR, M.: Citric Acid Fermentation and Temperature 97 Fig.1. Fermentation No. 1 (32 C) Fig. 2. Fermentation No. 3 (26.5 C) Fermentations at Temperatures around 26 C Fermentations were run with glucose concentrations of g/l (0.88 M) and g/l (0.85 M), respectively. Mean temperature was 26.5 C g/l (0.25 M) and 57.4 g/l (0.30 M) of citric acid were formed requiring a fermentation time of 322 (323) hours.

4 98 Acta Biotechnol. 23 (2003) 1 Residual sugar at the end of the fermentations was 0.14 and 0.10 M, respectively. The corresponding space-time yields accounted for 0.15 and 0.18 g/l. h, which is about 1 /4 the productivity at 32 C. The yields were found to be 35.8 and 42.8%. It may be calculated that (again considering biomass formation requiring sugar amounts of 0.14 M and 0.13 M, respectively) as much as about 48 and 44%, respectively, of the carbohydrate source utilised has to be considered as being wasted. The course of one of the fermentations is shown in Fig. 2. When the temperature was only lowered to about 28 C (no diagram), effects were similar but not as pronounced as described above. The same pertains to fermentations applying lower sugar concentrations. Such experiments, however, are less significant in terms of industrial practice. All the relevant data of these experiments are shown in Tab. 1. Experiments with Variation of Temperatures In order to scrutinise the previous results, experiments were conducted, in which the initial temperature was held at 32 C for periods of about 100 hours, and was subsequently lowered to 26.5 C, and held at this temperature for about 100 hours or more. The result of a typical experiment is shown in Fig. 3. The diagram clearly shows that concomitant with the change of temperature, the curve of sugar consumption for citric acid + biomass levels off rather sharply, displaying the resulting waste of the carbohydrate source as effected by the change of temperature to lower values. Again, this effect was induced and slightly less pronounced when temperature was only lowered to 28 C. Tab.1. Results of fermentations at different temperatures Parameters Number of fermentation Temperature [ C] Initial glucose [g/l] Final glucose = Residual glucose [g/l] Fermentation period [h] Glucose consumption [g/l] Citric acid [g/l] Space-time yield [g/l. h] Citric acid yield (based on glucose consumption) [%] Biomass [g d.wt./l] Glucose consumption for citric acid [g/l] Glucose consumption for biomass [g/l] Wasted glucose [g/l] Wasted glucose (based on sugar consumption) [%]

5 SELAHZADEH, R.M.,ROEHR, M.: Citric Acid Fermentation and Temperature 99 Fig. 3. Fermentation No. 8 (32 C and reduction to 26 C) Discussion The results presented clearly demonstrate the importance of temperature control in citric acid fermentation. Foremost, decreasing the temperature by several degrees causes an appreciably drastic decrease in the fermentation rate, as may be seen upon observing the course of sugar consumption. Most striking, however, is the metabolic effect showing that the system is wasting large amounts of the carbon source at these lower temperatures, apparently as a consequence of an increased respiratory activity. It should be mentioned that only negligible amounts of oxalic acid (below 0.1 g/l) and even lower amounts of other acids (e.g. traces of gluconic acid) could be detected by HPLC during the experiments. The results presented in Fig. 3 illustrate that the reaction of the system is rather prompt. Needless to say that the economic importance of this wasting metabolism is considerable, especially in view of the fact that modern citric acid industry uses rather large fermentation vessels with relatively simple cooling systems (cf. e.g. [5]). Our records do not permit exact statements, but it appears that increasing the temperature of a fermentation run that had been started at lower temperatures, e.g C, to e.g. 32 C, does not significantly react in the opposite way as would likely be expected. Received 24 October 2001 Received in revised form 12 August 2002 Accepted 17 September 2002

6 100 Acta Biotechnol. 23 (2003) 1 References [1] MIRMINACHI, F.,ZHANG, A., ROEHR, M.: Citric acid fermentation and heavy metal ions. I. Effects of iron, manganese and copper. Acta Biotechnol. 22 (2002), [2] ZHANG, A., ROEHR, M.: Citric acid fermentation and heavy metal ions. II. The action of elevated manganese ion concentrations. Acta Biotechnol. 22 (2002), [3] ZHANG, A.,ROEHR, M.: Effects of varied phosphorus concentrations on citric acid fermentation by Aspergillus niger. Acta Biotechnol. 22 (2002), [4] MUTZALL, K.: Modellierung von Bioprozessen. Hamburg: Behr s Verlag, 1994, [5] ROEHR, M.,KUBICEK, C.P.,KOMINEK, J.: Citric Acid. In: Biotechnology, 2 nd ed. (REHM, H.-J., REED, G.,PÜHLER, A.,STADLER, P., eds.). Vol. 6 (ROEHR, M., ed.). Weinheim: VCH, 1996, Erratum We regret that, unfortunately, an error occurred in the article ZHANG, A.,ROEHR,M.: Citric Acid Fermentation and Heavy Metal Ions II. The Action of Elevated Manganese Ion Concentrations published in Acta Biotechnol 22 (2002) 3 4. The caption of Fig. 6 should read Fig. 6. Poor fermentation at manganese concentration of 10 µg/l