Co-cultivation of filamentous microorganisms in the presence of aluminum oxide microparticles

Abstract

In the present work, the approaches of submerged co-cultivation and microparticle-enhanced cultivation (MPEC) were combined and evaluated over the course of three case studies. The filamentous fungus Aspergillus terreus was co-cultivated with Penicillium rubens, Streptomyces rimosus, or Cerrena unicolor in shake flasks with or without the addition of aluminum oxide microparticles. The influence of microparticles on the production of lovastatin, penicillin G, oxytetracycline, and laccase in co-cultures was compared with the effects recorded for the corresponding monocultures. In addition, the quantitative analyses of morphological parameters, sugars consumption, and by-products formation were performed. The study demonstrated that the influence of microparticles on the production of a given molecule in mono- and co-culture may differ considerably, e.g., the biosynthesis of oxytetracycline was shown to be inhibited due to the presence of aluminum oxide in "A. terreus vs. S. rimosus" co-cultivation variants but not in S. rimosus monocultures. The differences were also observed regarding the morphological characteristics, e.g., the microparticles-induced changes of projected area in the co-cultures and the corresponding monocultures were not always comparable. In addition, the study showed the importance of medium composition on the outcomes of MPEC, as exemplified by lovastatin production in A. terreus monocultures. Finally, the co-cultures of A. terreus with a white-rot fungus C. unicolor were described here for the first time. KEY POINTS: • Aluminum oxide affects secondary metabolites production in submerged co-cultures. • Mono- and co-cultures are differently impacted by the addition of aluminum oxide. • Effect of aluminum oxide on metabolites production depends on medium composition.

Keywords: Aspergillus terreus; Co-culture; Laccase; Lovastatin; Oxytetracycline; Penicillin.

Fig. 1

Influence of aluminum oxide (AO) on the morphology of Aspergillus terreus and Penicillium rubens grown in mono- and co-cultures. Microscopic images of A. terreus monoculture, P. rubens monoculture, and the “A. terreus vs. P. rubens” co-culture with and without the addition of AO are shown. The images were obtained after 24 h of cultivation. In all investigated variants, the supplementation with AO resulted in the decrease of pellet size compared to the non-AO counterparts. The medium used for the cultivation contained glucose (10 g L⁻¹), lactose (30 g L⁻¹), yeast extract (6 g L⁻¹), KH₂PO₄ (1.51 g L⁻¹), phenylacetic acid (0.5 g L⁻¹), biotin (0.04 mg L⁻¹), and salts. At least 45 objects were analyzed for each experimental variant, the selected examples are depicted here

Fig. 2

Influence of aluminum oxide (AO) on the values of morphological parameters determined for Aspergillus terreus and Penicillium rubens grown in mono- and co-cultures. Results of quantitative morphological analysis of pellets developed in the A. terreus monoculture, P. rubens monoculture and the “A. terreus vs. P. rubens” co-culture with and without the addition of AO microparticles are shown. The values of projected area (a), elongation (b), and roughness (c) parameters are presented as the mean value ± standard deviation with the average number of analyzed objects (n) equal to 45. The two-sample t-test (with a significance level of α = 0.05) was applied to verify if the results obtained for the AO variants differed significantly from their non-AO counterparts. *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001, ns, not significant

Fig. 3

Influence of aluminum oxide (AO) on the titers of secondary metabolites and the levels of carbon substrates in the mono- and co-cultures of Aspergillus terreus and Penicillium rubens. Time courses of lovastatin (a), penicillin G (b), glucose (c), and lactose (d) levels in the A. terreus monoculture and/or or P. rubens monoculture and the “A. terreus vs. P. rubens” co-culture with and without the addition of AO microparticles are shown. The concentration values are presented as “mean ± standard deviation” (n = 3). No traces of lovastatin or penicillin G were detected in 24 h of the cultivation run. The production of penicillin G was confirmed solely in P. rubens monocultures. The two-sample t-test (with a significance level of α = 0.05) was applied to verify if the results obtained for the AO variants differed significantly from their non-AO counterparts. *p ≤ 0.05, ***p ≤ 0.001, ns, not significant

Fig. 4

Influence of aluminum oxide (AO) on the morphology of Aspergillus terreus and Streptomyces rimosus grown in mono- and co-cultures. Microscopic images of A. terreus monoculture, S. rimosus monoculture and the “A. terreus vs. S. rimosus” co-culture with and without the addition of AO microparticles are shown. The images were obtained after 24 h of cultivation. The supplementation with AO resulted in the decrease of pellet size of A. terreus compared to the non-AO counterparts, whereas in the S. rimosus monocultures and the co-cultures this effect was not observed. The medium used for the cultivation contained glucose (20 g L⁻¹), lactose (20 g L⁻¹), yeast extract (5 g L⁻¹), KH₂PO₄ (1.51 g L⁻¹), biotin (0.04 mg L⁻¹), and salts. At least 45 objects were analyzed for each experimental variant, the selected examples are depicted here

Fig. 5

Influence of aluminum oxide (AO) on the values of morphological parameters determined for Aspergillus terreus and Streptomyces rimosus grown in mono- and co-cultures. Results of quantitative morphological analysis of pellets developed in the A. terreus monoculture, S. rimosus monoculture, and the “A. terreus vs. S. rimosus” co-culture with and without the addition of AO microparticles are shown. The values of projected area (a), elongation (b), and roughness (c) parameters are presented as the mean value ± standard deviation with the average number of analyzed objects (n) equal to 45. The two-sample t-test (with a significance level of α = 0.05) was applied to verify if the results obtained for the AO variants differed significantly from their non-AO counterparts. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, **** p ≤ 0.0001, ns, not significant

Fig. 6

Influence of aluminum oxide (AO) on the titers of secondary metabolites and the levels of carbon substrates in the mono- and co-cultures of Aspergillus terreus and Streptomyces rimosus. Time courses of lovastatin (a), oxytetracycline (b), glucose (c), and lactose (d) levels in the A. terreus monoculture and/or S. rimosus monoculture and the “A. terreus vs. S. rimosus” co-culture with and without the addition of AO microparticles are shown. The concentration values are presented as “mean ± standard deviation” (n = 3). No traces of lovastatin or oxytetracycline were detected in 24 h of the cultivation run. The production of lovastatin was confirmed solely in A. terreus monocultures. The two-sample t-test (with a significance level of α = 0.05) was applied to verify if the results obtained for the AO variants differed significantly from their non-AO counterparts. *p ≤ 0.05, **p ≤ 0.01, ns, not significant

Fig. 7

Influence of aluminum oxide (AO) on the morphology of Aspergillus terreus and Cerrena unicolor grown in mono- and co-cultures. Microscopic images of A. terreus monoculture, C. unicolor monoculture and the “A. terreus vs. C. unicolor” co-culture with and without the addition of AO microparticles are shown. The images were obtained after 24 h of cultivation. The presented examples illustrate the increase in pellet size due to the addition of AO in all tested variants. The medium used for the cultivation contained glucose (10 g L⁻¹), yeast extract (2 g L⁻¹), KH₂PO₄ (0.47 g L⁻¹), L-asparagine (1.5 g L⁻¹), thiamine (50 µg L⁻¹), and salts. At least 45 objects were analyzed for each experimental variant, the selected examples are depicted here

Fig. 8

Influence of aluminum oxide (AO) on the values of morphological parameters determined for Aspergillus terreus and Cerrena unicolor grown in mono- and co-cultures. Results of quantitative morphological analysis of pellets developed in the A. terreus monoculture, C. unicolor monoculture, and the “A. terreus vs. C. unicolor” co-culture with and without the addition of AO microparticles are shown. The pellets of C. unicolor at 96 h became too large to conduct the microscopic observations (as in the work of Antecka et al. 2016b), so the morphological investigation in the “A. terreus vs. C. unicolor” case study was conducted until 72 h of the run. The values of projected area (a), elongation (b), and roughness (c) parameters are presented as the mean value ± standard deviation with the average number of analyzed objects (n) equal to 45. The two-sample t-test (with a significance level of α = 0.05) was applied to verify if the results obtained for the AO variants differed significantly from their non-AO counterparts. **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, ns, not significant

Fig. 9

Influence of aluminum oxide (AO) on the titers of lovastatin and laccase and the levels of glucose in the mono- and co-cultures of Aspergillus terreus and Cerrena unicolor. Time courses of lovastatin (a), laccase (b), and glucose (c) levels in the A. terreus monoculture and/or C. unicolor monoculture and the “A. terreus + C. unicolor” co-culture with and without the addition of AO microparticles are shown. The concentration values are presented as “mean ± standard deviation” (n = 3). No traces of lovastatin or laccase activity were detected in 24 h of the cultivation run. As shown in (c), glucose was no longer detectable after 144 h of the run in all the tested variants. The cultivation was performed for 216 h as this time is required to achieve relatively high levels of laccase (Antecka 2016b). The two-sample t-test (with a significance level of α = 0.05) was applied to verify if the results obtained for the AO variants differed significantly from their non-AO counterparts. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ns, not significant