Alginate and galactomannan-derived oligosaccharides enhanced the production of penicillin G when added to stirred tank reactor cultures of Penicillium chrysogenum. The addition of oligomannuronate and oligoguluronate blocks increased penicillin G yield by 47% and 49%, respectively. The effect of mannan oligosaccharides was found to be more pronounced with 69% higher yield than the control cultures. The maximum increase in the average specific productivity of the oligosaccharide augmented cultures was 55% after addition of mannan oligosaccharides. In addition, a difference was observed in all cases in the accumulation pattern of the intermediate of penicillin biosynthesis, δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine.
In recent years, the function of oligosaccharides as elicitors has been well studied in production of secondary metabolites by plant cell cultures [1, 2]. These studies were focused firstly on the biosynthetic processes related to plant defense mechanisms and gradually extended into the production of bioactive substances for pharmaceutical, agricultural and industrial chemicals.
Alginate oligosaccharides have been implicated in the elicitation of secondary metabolites in cultures of Lithospermum erythrorhizon. Other secondary metabolites produced in increased yields through the influence of carbohydrate fragments in plant cell cultures include indole alkaloids such as acridone [4, 5], the phytoalexin (plant antibiotic) pisatin  and steroids such as solasodine and disogenin [7, 8]. Chitinase activity has been reported to be stimulated in plants contacted with chitosan and chitosan oligosaccharides . Messiaen et al.  have achieved prolonged increase in cytosolic calcium in carrot protoplasts by use of oligogalacturonides obtained from fungal cell walls. In the studies carried out on fungal cell cultures, chitosan was found to induce profound ultrastructural and morphological changes in the filamentous fungus Fusarium oxysporum. Yonemoto et al.  and Tomoda et al.  also reported growth promoting effects of alginate oligosaccharides on plants. In animals, chitin oligosaccharides have been shown to have anti-tumour activity  and chitosan oligosaccharides have reported enhanced serum lysozyme activity . In cultures of Bifidobacteria, alginate oligosaccharides, amongst other oligosaccharides tested, accelerated growth when added as a supplement to the culture medium .
Many commercially important secondary metabolites have been produced by microbial cultures. Given the notable role of fungal cultures in the production of secondary metabolites such as antibiotics, it is clearly of practical as well as theoretical interest to seek the evidence of enhancement of secondary metabolite yields in fungal cultures. One of the most extensively studied microbial fermentation processes has been the production of penicillin by the filamentous fungus Penicillium chrysogenum. The production of penicillin is well characterised and therefore forms a suitable model system for studying the effects of oligosaccharides on the physiology of secondary metabolism in fungi.
We have studied the effect of alginate oligosaccharides in shaken flask cultures of P. chrysogenum. In a previous paper, we reported that the addition of alginate oligosaccharides oligomannuronate (OM) and oligoguluronate (OG) resulted in an increase of 36 and 13%, respectively, in the yield of penicillin G . In this study, we examine the enhancement effect of alginate oligosaccharides and mannan oligosaccharides (MO) in stirred tank reactors. Fermentation profiles of P. chrysogenum batch fermenter cultures after the addition of alginate oligosaccharides showed that the oligosaccharides have a more pronounced effect on penicillin yields in the controlled environment of a fermenter than shaken flask cultures. The mannan oligosaccharides were found to have the highest enhancement effect among the oligosaccharides examined.
2Materials and methods
2.1Strains and culture medium
Penicillium chrysogenum strain P2 was maintained on solid agar slopes of glycerol and molasses medium . Semi-defined media were used for growth and penicillin production. The growth medium contained: 20 g sucrose, 10 g lactose, 5 g mycological peptone, 13 g (NH4)2SO4, 3 g KH2PO4, 0.5 g Na2SO4, 0.55 g EDTA, 0.25 g MgSO4·7H2O, 0.05 g CaCl2·2H2O, 0.25 g FeSO4·7H2O, 0.02 g MnSO4·4H2O, 0.02 g ZnSO4·7H2O, and 0.005 g CuSO4·5H2O in 1000 ml of distilled water. The pH of the growth medium was adjusted to 6.8 with KOH before sterilisation. The semi-defined production medium was as above but contained 100 g of lactose and 1 g of mycological peptone. For penicillin G production phenylacetic acid (PAA) was added to 24-h old cultures to make a final concentration of 1.5 g l−1. PAA level was kept between 0.5 and 1.5 g l−1 in the fermenter cultures by intermittent addition of a concentrated solution when necessary.
Spore suspensions of strains of P. chrysogenum P2 were inoculated into 2-l flasks containing 20% v/v sterile semi-defined growth medium to give a final concentration of 5×105 spores ml−1. The flasks were then incubated at 26°C in an orbital shaker at 200 rpm for 48 h to prepare inoculum for fermentation in a stirred tank reactor (STR).
Fermentations were carried out in 5-l stirred tank reactors (Inceltech, Reading, UK) with 4 l working volume. The reactors were baffled and medium was agitated by two Rushton turbine impellers located 12 cm apart on the drive shaft. Air was sparged into the medium at a flow rate of 0.5 vvm and the stirrer speed was set at 600 rpm. The pH of the culture was maintained between 6.7 and 6.9 by the controlled addition of 2 M aqueous ammonia and 2 M sulfuric acid as required and the temperature was 26°C. Foaming was controlled by addition of 1 ml of antifoam (Henkel-Nopco Foamaster TDB-1) per liter medium before sterilisation. In situ sterilisation of the medium was carried out at 121°C and 1.01 atm for 15 min.
The medium in the STR was inoculated aseptically with a 48-h old inoculum culture of P. chrysogenum P2 (10% v/v). Samples were removed at regular intervals and filtered for dry cell weight analysis. Filtrates were kept at 0°C for later analyses.
2.4Addition of alginate and mannan oligosaccharides
OM and OG blocks with an average degree of polymerisation (DP) of 10 were prepared as described previously . The blocks were dissolved in distilled water and pH was adjusted to 6.8. The solutions were autoclaved at 121°C and 1.01 atm for 15 min. Appropriate amounts of OM and OG block solutions were added to cultures of P. chrysogenum at 48 h of growth in semi-defined production medium.
Mannan oligosaccharides were prepared from locust bean gum (LBG) by enzymatic hydrolysis with Gamanase enzyme mixture (Novo-Nordisk Ltd, Denmark). LBG (1 g) was dissolved in 50 ml distilled water, heated to 80°C, and 0.1 ml of Gamanase was added. The reaction was carried out for 5 min and quenched by heating to 100°C. The hydrolysis products were analysed by thin-layer chromatography and the mannan oligosaccharides were separated by gel-filtration chromatography. Mannan oligosaccharides having a DP of 7 were added to the cultures in a similar way to OM and OG blocks.
2.5Analysis of samples for growth, penicillin G and ACV
Dry cell weight (DCW) was determined by collecting 20-ml samples which were filtered through preweighed Whatman No. 1 filter paper and washed thoroughly with water and then dried at 80°C to a constant weight. Penicillin G and the precursor tripeptide δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine (ACV) concentrations in each culture were determined by an HPLC method . Two fermentations were carried out in each case and samples in duplicate were used in all assays.
3Results and discussion
A series of fermentations were carried out in stirred-tank reactors using alginate or mannan oligosaccharides to investigate the growth profile of P. chrysogenum P2 together with penicillin G production. Based on our previous study  OM and OG blocks with a DP of 10 and concentration of 25 mg l−1 were used for penicillin G enhancement in the reactors. Mannan oligosaccharides were also used at a concentration of 25 mg l−1 but with a DP of 7, which was shown to have the optimal effect in shaken flask cultures . Using these oligosaccharides, the physiology of P. chrysogenum P2 cultures was compared with those of control fermentations (without oligosaccharide addition) with respect to growth and penicillin G production and accumulation of the penicillin intermediate (ACV).
A representative fermentation profile is reported for each case. Duplicate samples from the fermentations showed good agreement with a coefficient of variation CV≤5.
3.1Addition of alginate oligosaccharides to the cultures
In the cultures complemented with OM or OG blocks 47 and 49% increases, respectively, in penicillin G yields were observed when compared with control cultures without the oligosaccharides (Fig. 1). The biomass and penicillin G production profiles were similar for OG and OM supplemented cultures. In OM supplemented cultures the maximum penicillin G concentration was 4 g l−1 corresponding to an increase in penicillin G production of 43% over that obtained in the control fermentation. In OG added cultures penicillin G production was 55% higher than the control reaching 4.4 g l−1. OM and OG blocks are structurally similar to each other not only by being hexose oligomers but also by being composed of modified monosaccharide units in which the CH2OH group has been oxidised to COO− (carboxylate groups). Their mechanism of action to cause enhancements in penicillin G production in cultures of P. chrysogenum is expected to be alike because of their structural similarities.
The penicillin precursor ACV showed similar profiles in cultures supplemented both with OM and OG blocks but the accumulation of ACV in the culture was greater in the case of cultures with OG blocks added than in cultures with OM blocks added (Fig. 2). This could be due to the increased activity of ACV synthase as a result of the presence of OG blocks that could have interaction with the regulatory process of penicillin biosynthesis. OG blocks may lower calcium activity by chelating calcium ions [20, 21]; this can affect the production of ACV synthase. Although OG and OM blocks have similar structures, their conformational differences seem to result in an increased activity of ACV synthase in the case of OG blocks. The penicillin G production was found to be similar in OG or OM supplemented cultures. The conversion of ACV to penicillin G may be rate limiting, resulting in accumulation of ACV leading to the production of a similar amount of penicillin G in the presence of OM and OG blocks.
3.2Addition of mannan oligosaccharides to the cultures
Growth and penicillin G production profiles were similar to those of the cultures with OM and OG blocks added but the concentration of biomass in MO added cultures was the lowest (15% less than the control). The effect of MO on cell metabolic activity seems to be both on biomass production and product formation resulting in highest yield when compared to other cultures. However, the overproduction was more pronounced and addition of mannan oligosaccharides resulted in 69% higher penicillin G yields than the control cultures without oligosaccharides (Figs. 1 and 3). Mannan oligosaccharides have structural similarities to alginate oligosaccharides. The mannan oligosaccharide chain consists of β-1,4 linked mannose units, while OM blocks consist of β-1,4 linked mannuronate units. These oligosaccharides belong to the same carbohydrate conformational group. It could be speculated that P. chrysogenum cell wall provides carbohydrate recognition sites containing mannose receptors. When the receptors meet mannan oligosaccharides of specific size and conformation, they respond by further activating (compared to control) enzyme(s) involved in the biosynthesis of penicillin, resulting in increased penicillin G production. Lower levels of ACV are observed in the cultures supplemented with mannan oligosaccharides than in cultures with OM or OG added (Fig. 2). This could be due to a more efficient conversion of ACV to penicillin G resulting in higher penicillin G production.
The average penicillin G production rates (specific and volumetric) during fermentations are shown in Table 1. Compared with the control fermentations, the cultures with added oligosaccharides showed higher penicillin productivities in all cases studied. The maximum increase in specific productivity was in the case of mannan oligosaccharide addition (55%) while OG had the highest effect in terms of volumetric productivity (34%). Addition of oligosaccharides did not have a significant effect (changes were within 10%) on the maximum specific growth rate when compared with control culture (Table 1).
Table 1. Maximum specific growth rates (μmax; h−1) and average penicillin G productivities (specific productivity, qpen: mg g−1 h−1; volumetric productivity, Qpen: mg l−1 h−1) of the fermentation cultures
μmax (h−1) (0–48 h)
qpen (mg g−1 h−1) (24–144 h)
Qpen (mg l−1 h−1) (24–144 h)
In agreement with our previous paper , penicillin G yields showed similar trends in their response to oligosaccharide addition but the enhancement by oligosaccharides was greater in the more controlled environment of the STR (Fig. 3). In shaken flask cultures, oxygen limitation is a problem and has profound effects in the fermentations of aerobic fungal cultures. Such a limitation may also affect the way in which carbohydrate fragments interact with regulatory signals governing penicillin G production.