Cocultivation of White-Rot Fungi and Microalgae in the Presence of Nanocellulose

ABSTRACT Cocultivation of fungi and algae can result in a mutualistic or antagonistic interaction depending on the species involved and the cultivation conditions. In this study, we investigated the growth behavior and enzymatic activity of two filamentous white-rot fungi (Trametes versicolor and Trametes pubescens) and two freshwater algae (Chlorella vulgaris and Scenedesmus vacuolatus) cocultured in the presence of TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl radical) oxidized cellulose nanofibrils (CNF) and cellulose nanocrystals (CNC). The growth of fungi and algae was studied in liquid, agar medium, and 3D-printed nanocellulose hydrogels. The results showed that cocultures grew faster under nutrient-rich conditions than in nutrient-depleted conditions. Key cellulose-degrading enzymes, including endoglucanase and laccase activities, were higher in liquid cocultures of T. versicolor and S. vacuolatus in the presence of cellulose compared to single cultures of fungi or algae. Although similar results were observed for cocultures of T. pubescens and C. vulgaris, laccase production diminished over time in these cultures. Fungi and algae were capable of growth in 3D-printed cellulose hydrogels. These results showed that cellulase enzyme production could be enhanced by cocultivating white-rot fungi with freshwater algae under nutrient-rich conditions with TEMPO-CNF and CNC. Additionally, the growth of white-rot fungi and freshwater algae in printed cellulose hydrogels demonstrates the potential use of fungi and algae in hydrogel systems for biotechnological applications, including biofuel production and bio-based fuel cell components. IMPORTANCE Depending on the conditions used to grow fungi and algae in the lab, they can interact in a mutually beneficial or negative way. These interactions could stimulate the organisms to produce enzymes in response to the interaction. We studied how wood decay fungi and freshwater algae grew in the presence and absence of cellulose, one of the basic building blocks of wood. How fungi and algae grew in 3D-printed cellulose hydrogels was also tested. Our results showed that fungi and algae partners produced significantly larger amounts of enzymes that degraded cellulose when grown with cellulose than when grown alone. In addition, fungi and algae were shown to grow in dense nanocellulose hydrogels and could survive the shear conditions during gel structuring while 3D-printing. These cultures could potentially be applied in the biotech industry for applications like energy production from cellulose, biofuel production, and bioremediation of cellulose material.


Sample preparation and SEM imaging of T. versicolor 159 and S. vacuolatus
T. versicolor and S. vacuolatus were inoculated in 70 mL of 1:1 media mixture supplemented with 1g TEMPO-oxidized CNF (1.2 wt%) and sulfuric acid hydrolyzed CNC (15 wt%) giving a final concentration of CNF and CNC 0.017 wt% and 0.2 wt% respectively.
Cultures were initially shaken for 2 days at 120 rpm to promote the growth of the coculture and then left to stand at ambient room temperature. Co-cultures were grown under an LED light (as less light was available for growth during the fall) (Hama Stick; blue-red setting, 9 hour cycle). Over time, the co-culture formed a mat-like structure at the surface of the liquid medium. This structure was carefully transferred to a petri dish, frozen by the addition of liquid nitrogen, and lyophilised overnight (Alpha 3-4 LSCbasic). A small portion of the lyophilised sample (approximately 1 cm x 2 cm) was cut, coated with a 7nm layer of platinum (Bal-Tec Med 020), and assessed via SEM (FEI QUANTA 650FEG ESEM).

Growth of co-cultures inside printed inks
To monitor the growth of co-cultures inside printed inks using confocal microscopy, TEMPO-oxidized CNF (1.2 wt%) and sulfuric acid hydrolyzed CNC (10 wt%)) were prepared as described above, except that after the addition of the malt extract, the ink was orbital mixed at 1500 rpm for 1 min and 2300 rpm for 4 min and stored at 4°C in the dark until cell transfer. Sulfuric acid hydrolyzed CNC (20 wt%) was prepared using 1:1:SK:ME by combining 16.44 g CNC and 80 mL of 1:1:SK:ME, mixed with a spatula and orbital mixed at 1500 rpm 1 min and 2300 rpm for 4 min. The ink was stored for 2-3 days at 4°C. Next, 40 g of the ink was orbital mixed for 1 min before autoclaving as described above. During cell transfer, 10 5 C. vulgaris cells, grown in Sueoka`s high salt medium were transferred to 40 g of ink by diluting cells in 1X PBS solution to a final volume of 100 μL. The ink was mixed by orbital mixing the ink at 1500 rpm for 1 min and 2300 rpm at 4 min. Algae cell counts were determined using a Neubauer chamber and a Zeiss Axioplan optical microscope with trans-illumination and an Epiplan NEOFLUR 10X objective. T. pubescens 220 was transferred to inks by scraping off 0.02 g of mycelium from a 2% MEA plate. The sample was transferred to a 50 mL sterile Falcon tube and 5 mL of 1X PBS added along with sterile glass beads (Sigma, 2 mm diameter). The suspension was mixed (Vortex Genie 2, Scientific Industries, 2 min, level 10) until the fibers broke apart. 2 mL of this suspension was added to the ink and orbital mixed as above before storing the ink at 4°C in the dark.

Confocal laser scanning microscopy (CLSM) images of printed inks
TEMPO-oxidized CNF and CNC printed inks containing co-cultures were prepared by

Confocal laser scanning microscopy (CLSM) images of co-cultures
For confocal microscopy fungal pellets with attached algae that were growing for 23 days in the 1:1 ME:SK media mixture, were cut into pieces with a sterile scalpel.