T. reesei RUT-C30 (RFP1) (a red fluorescent protein labeled clone (7) of the standard RUT-C30 strain obtained from American Type Culture Collection ATTC 56765) was propagated at 30 °C on potato extract glucose agar medium (Roth, Karlsruhe, Germany) containing 40 mL/L of carrot juice (BIOBIO, Marken-Discount AG & Co. KG, Germany). P. verruculosum M28-10, kindly gifted by Dr. Gerhard Kerns (Saxon Institute for Applied Biotechnology, Leipzig, Germany), was propagated at 30 °C on medium containing 10 g/L malt extract (Difco; Becton, Dickinson and Company, USA), 40 mL/L carrot juice, 10 g/L wheat bran (Alnatura, Darmstadt, Germany), 10 g/L α-cellulose (Sigma-Aldrich, St. Louis, USA), 30 g/L agar (Difco; Becton, Dickinson and Company, USA). Spores were harvested from agar plates using 10 mL 0.01% (v/v) Tween 80 solution and washed twice with bi-distilled water. The spore concentration was determined in a Neubauer-Improved counting chamber (Superior Marienfeld, Lauda-Königshofen, Germany), adjusted to 109 spores/mL and stored at 4 °C. This 1000 × concentrated stock was used for inoculation all experiments.
U. maydis Δcyp3 ΔPria1::Petef Δfuz7 PetefmttA is a genetically engineered variant of U. maydis MB215 with enhanced itaconate production, reduced by-product formation, and stabilized morphology (20). This strain was propagated at 30 °C on yeast extract peptone dextrose (YPD) agar plates. Liquid overnight cultures of U. maydis were grown from single agar plate colonies at 30 °C in YPD medium. For inoculation of the experiments, YPD cultures were washed twice with bi-distilled water and used to inoculate the experiments to the final OD as indicated in the figure captions. For the initial SSF and batch CBP experiments, freshly prepared aqueous cell suspensions were used for inoculation. For the fed-batch CBP experiment, the same aqueous cell suspension stock was used for all tested inoculation delays and stored in an ice bath.
Media and cultivation
All experiments were performed in 250 mL Erlenmeyer flasks with 25 mL filling volume at 30 °C, 200 rpm and 50 mm shaking diameter.
The itaconic acid production medium used in the initial SSF experiment was adopted from Geiser et al. (49). Because the added cellulase containing fermentation supernatants of the cellulase producers contained already residual NH4+, NH4Cl was omitted. The medium contained 0.2 g/L MgSO4·7H2O, 0.01 g/L FeSO4·7H2O, 0.5 g/L KH2PO4, 1 mL/L vitamin solution, 1 mL/L trace element solution, and as buffer 19.5 g/L (100 mM) 2-(N-morpholino) ethanesulfonic acid (MES) or CaCO3 as indicated in the figure captions. The pH of the MES stock solution was adjusted to 6.5 with NaOH. The trace element solution contained 15 g/L EDTA, 4.5 g/L ZnSO4·7H2O, 1 g/L MnCl2·4H2O, 0.3 g/L CoCl2·6H2O, 0.3 g/L CuSO4·5H2O, 0.4 g/L Na2MoO4·2H2O, 4.5 g/L CaCl2·2H2O, 3 g/L FeSO4·7H2O, 1 g/L H3BO3, and 0.1 g/L KI. The vitamin solution contained 0.05 g/L D-biotin, 1 g/L D-calcium panthotenate, 1 g/L nicotinic acid, 25 g/L myo-inositol, 1 g/L thiamine hydrochloride, 1 g/L pyridoxol hydrochloride and 0.2 g/L para-aminobenzoic acid. The medium was prepared as a 1.43 × concentrated stock solution. The solution was filtered through a 0.2 µm filter for sterilization. Before the experiment, the 1.43 × concentrate was diluted to its original concentration by addition of sterile bidest water and sterile filtered cellulase containing fermentation supernatants. The cellulase containing fermentation supernatants of T. reesei RUT-C30 (RFP1) and P. verruculosum M28-10 were produced in a stirred tank fermentation as described in Supplementary File S7. The necessary amount of cellulose was directly weighted into empty Erlenmeyer flasks (3 g Sigmacell or 3 g α-cellulose) and steam-sterilized at 121 °C as powder before the liquid medium was added. Both types of cellulose were purchased from Sigma-Aldrich (St. Louis, USA). For the single feeding event during the SSF cultivation, 1.5 g the corresponding cellulose was steam-sterilized as powder and added separately to each Erlenmeyer flask.
The medium for the co-culture CBP was based on a medium published by Pakula et al., which was modified for co-culture compatibility with A. terreus, U. maydis and T. reesei, enabling both itaconic acid and cellulase production by the respective organisms (50, 51). The final medium consisted of (NH4)2SO4 7.6 g/L, KH2PO4 0.8 g/L, MgSO4·7H2O 0.5 g/L, CaCl2·2H2O 0.23 g/L, NaCl 0.05 g/L, 5 g/L CaCO3, glucose 5 g/L, α-cellulose 30 g/L, peptone ex casein 2 g/L (N-Z-Amine® AS, Carl Roth, Karlsruhe, Germany), Tween 80 0.02% (v/v), trace element solution 2.5 mL/L. The main solution without trace elements and cellulose was always prepared as a 2 × concentrated stock solution that was set to pH 5.5 with 5 M NaOH. The solution was filtered through a 0.2 µm filter for sterilization. Before the experiment, the 2 × concentrate was diluted to its original concentration by addition of sterile bidest water and other supplementing solutions such as trace elements or glucose. The trace element solution (400 × concentrated) had the following composition: citric acid 180 g/L, Fe2(SO4)3 2.29 g/L, ZnSO4·7H2O 16 g/L, CuSO4 2.05 g/L, MnSO4·7H2O 1.6 g/L, H3BO3 0.8 g/L, CoCl2·6H2O 2.71 g/L.
For the batch CBP experiment, T. reesei was cultivated at 200 rpm, 50 mm shaking diameter and 30 °C in three 1L Erlenmeyer flasks with 100 mL filling volume for 7 days in the described co-culture medium buffered with 33 g/L CaCO3. Thereafter, the three cultures were pooled and phosphate and ammonium content of the culture was measured. Residual NH4+ was equivalent to 1.2 g/L NH4Cl and was not necessary to supplement before the inoculation of U. maydis since the NH4Cl concentration in itaconic acid production medium is only 0.8 g/L. Residual KH2PO4 was 0.18 g/L and was supplemented to a final concentration of 0.5 g/L to prevent preliminary phosphate limitation. For the CBP experiment, the T. reesei culture broth was distributed to three replicate Erlenmeyer flasks (25 mL each), for the corresponding SSF experiment, the residual T. reesei culture broth was sterile filtrated and also distributed into three replicate Erlenmeyer flasks (25 mL each). The necessary amount of cellulose and CaCO3 was directly weighted into the empty Erlenmeyer flasks and steam-sterilized at 121 °C as powder before the liquid was added. Thereby, the cultures were supplemented with 120 g/L α-cellulose and 33 g/L CaCO3.
For the fed-batch CBP, the KH2PO4 starting concentration in the co-culture medium was increased to 1.3 g/L to anticipate the KH2PO4 supplementation that was necessary in the batch CBP. CaCO3 was increased to 40 g/L.
The reference cultivation of U. maydis with 50 g/L glucose was performed in in the co-culture medium with only 1.5 g/L NH4SO4, 0.5 g/L KH2PO4, without cellulose and buffered with 40 g/L CaCO3.
1 mL Samples were withdrawn from the same Erlenmeyer flasks during the cultivation (no sacrifice flasks). The weight of the Erlenmeyer flasks was determined before every sampling to correct the measured variables for evaporation. When necessary, bi-distilled water was added to compensate for significant evaporation.
Phosphate was determined according a method for orthophosphate determination published by the United states EPA (52). Ammonium was determined according to a modified version of the Berthelot reaction (53). The protein concentration of the culture supernatant was determined by a Bradford assay (54) using Coomassie Plus™ assay reagent (Thermo Scientific, Waltham, USA) and bovine serum albumin as standard. The measurement procedure was performed according to the manufacturer’s manual for microtiter plates. Cellulase activity in the culture supernatant was measured by the standard filter paper activity (FPA) assay according to the method of Ghose (55) adapted by Xiao (56). The assay was performed in 60 µL reaction volume in 96-well conical bottom PCR plates.
Soluble sugars and metabolites including glucose, cellobiose, xylose as well as itaconic acid, citric acid, malic acid and succinic acid were determined via HPLC analysis. To dissolve potentially precipitated calcium itaconate, the broth was diluted 6 × with 0.5M HCl. After centrifugation of the fermentation samples (16,900 g; 10 min; 4 °C) and a second centrifugation step of the resulting supernatant (3,000 g; 10 min), the supernatant was analyzed by HPLC (Dionex HPLC UltiMate 3000, Thermo Scientific, Waltham, USA) at 65 °C using the following setup: Column: AMINEX Ion Exclusion HPX-87H, 300 × 7.8 mm (Bio-Rad Laboratories GmbH, Munich, Germany); detectors: Dionex™ Ultimate 3000 UV/VIS detector (Thermo Scientific, Waltham, USA) at 210 nm and RI-101 refractory index detector (Shodex, Munich, Germany); mobile phase: 5 mM sulfuric acid; flow rate: 0.7 mL/min.
For the standard determination of CDW in absence of cellulose particles, between 0.75 and 3 mL of sample were filled into pre-weighted conical bottom glass tubes and centrifuged (20 min, 3,000 g, 4 °C). The supernatant was carefully pipetted off for other analytic procedures. The resulting pellet was washed two times by resuspension in 10 mL dest. water with subsequent centrifugation (20 min, 3000 g, 4 °C) and careful removal of the water using a pipette connected via a collecting bottle to a vacuum pump. Thereafter, the washed pellet was dried overnight (at least 12 h) in a Speedvac device under vacuum at 40 °C and 300 g acceleration. Finally, the glass tube containing the dried pellet was weighed on a microbalance and the CDW was calculated by subtracting the empty weight of the tube.
For the determination of CDW and residual cellulose in samples containing cellulose particles, a modified version of the Updegraff method adapted by Ahamed & Vermette was used (57, 58). First, the total dry weight of all solids (TDW) was determined as described for the standard CDW determination above. The resulting dry pellet was then re-suspended in 3 mL of Updegraff reagent and incubated for 30 min in a boiling water bath with a marble on top of the glass tube to reduce evaporation. Thereby, the Updegraff reagent selectively dissolves the fungal biomass, leaving the cellulose intact. Updegraff reagent is a mixture of 10 mL conc. nitric acid and 100 mL 80% acetic acid. After the incubation, the suspension was mixed with 3 mL 96% ethanol to facilitate sedimentation of the cellulose and centrifuged (20 min, 3000 g, 4 °C). The resulting pellet was washed two times by resuspension in 10 mL dest. water with subsequent centrifugation (20 min, 3000 g, 4 °C) and careful removal of the water using a pipette connected via a collecting bottle to a vacuum pump. Thereafter, the pellet was washed with 1 mL 70% ethanol without subsequent resuspension and dried overnight (at least 12 h) in a Speedvac device under vacuum at 40 °C and 300 g acceleration. Finally, the glass tube containing the dried pellet was weighed on a microbalance and the weight of cellulose was calculated by subtracting the empty weight of the tube. The corresponding CDW was determined by subtracting the weight of cellulose from the TDW.
The offgas analysis was realized using a commercial Transfer-Rate Online Measurement (TOM) system (Kuhner, Birsfelden, Switzerland) equipped with a mass flow controller.
Online scattered light measurements
Online scattered light measurements were performed using the cell growth quantifier (CGQ) system (Aquila biolabs GmbH, Baesweiler, Germany).
Microscopy and U. maydis cell counting
T. reesei and U. maydis were microscopically discriminated by their differing cell wall composition. Therefore, samples were stained with a mixture calcofluor, which predominantly stains β-1,4-glucan and trypan blue, which predominantly stains chitin (59). The sample suspensions were first diluted 1/10 with bi-distilled water and then 1/10–1/20 with the following staining solution: 10 µg/mL trypan blue and 10 µg/mL calcofluor in 20 mM phosphate-citrate buffer pH 7.4. Finally, 11 µL of the diluted and stained suspension were pipetted into wells of a µ-Slide angiogenesis (ibidi GmbH, Gräfelfing, Germany) and analyzed at 10X magnification (Plan-Apochromat 10X objective with 1X tubelens optovar) by a ZEISS Axio Observer Z1 (Zeiss, Jena, Germany) inverted fluorescence microscope equipped with a Yokogawa CSU-X1 spinning disk unit. Calcofluor fluorescence was recorded with 405 nm laser excitation, RQFT 405/488/568/641 dichroitic mirror and BP 450/50 nm emission filter. Trypanblue fluorescence was recorded with 638 nm laser excitation, RQFT 405/488/568/641 dichroitic mirror and BP 690/50 nm emission filter. Per sample, a total of 8 different field of views were recorded. For extended focus depth, 5 slices covering a Z-dimension range of 32 µm were recorded and processed into a maximum projection image. Besides the differing morphology, U. maydis yeast cells showed stronger trypan blue fluorescence relative to T. reesei hyphae, which allowed manual counting of the yeast cells in the images.