S enhancement was observed in media containing 39 g/L sucrose and
S enhancement was observed in media containing 39 g/L sucrose and 16 g/L peptone at pH 3.8 [46]. Shake flask cultures of A. adeninivorans strains were analyzed using a device for online measurement of the respiration rates (RAMOS, respiratory RM-493 msds activity monitoring system) [47,48]. This device had previously been applied to the analysis of H. polymorpha cultures and to alternative platforms [49-52]. In a first series yeast minimal medium (YMM) was assessed. Prior to any practical experiment, YMM ammonium concentration of the standard medium was raised from 2.2 mmol N/g glucose to 4.6 mmol N/g glucose since theoretical material balancing revealed a severe lack of nitrogen with regard to the average nitrogen content of yeast. The culture broth was additionally supplemented with calcium and iron in higher concentrations as well asReporter vectorMOX(p)GFPFigure 2 pathway promoter-controlled GFP production in recombinant H. polymorpha strains Methanol Methanol pathway promoter-controlled GFP production in recombinant H. polymorpha strains. A. Screening of CBS4732-derived transformants producing GFP under control of the FMD promoter. Screening of 44 different H. polymorpha pC10-FMD (PFMD-GFP) clones cultured under different cultivation modes; screening experiments were performed in deep-wellplates for batch and in FeedBead96-plates for fed-batch cultivation. Gas permeable sealings were used as sterile closures. To reduce the evaporation during the cultivation, water-saturated air was used for gassing. To compare the results from the different cultivation modes an identical carbon source concentration of 16.63 g/L were applied to each fermentation (batch with glycerol, batch with glucose, fed-batch with glucose). The batch cultivations were stopped when the stationary growth phase (glycerol 21 h, glucose 16 h) was reached and the fed-batch cultivations were stopped when 16.63 g/L glucose were released from the FeadBeat96-plate (16 h). Dry cell weight (DCW) was calculated from optical density measurements in a PowerWave ?40 micro titer plate reader. Green Fluorescent Protein (GFP) was measured at 485 nm excitation and 520 nm emission wavelengths. Cultivation conditions: shaking frequency 400 rpm, shaking diameter 50 mm, temperature 37 , filling volumes 300 L per well; media: SYN6-MES with 16.63 g/L of different carbon sources. Carbon sources: batch mode with glycerol (black line), batch mode with glucose (grey bars), fed-batch mode with glucose (black bars); inocula ratio 1:30. B. GFP production in a DL-1-derived transformant under control of the MOX promoter. H. polymorpha transformants producing GFP under control of MOX promoter were cultured for 12 h on YP medium containing 10 g/L methanol (YP+1 M) or 0.5 g/L glucose (YP+0.05 D) and then analyzed by confocal microscopy.Page 5 of(page number not for citation purposes)Microbial Cell Factories 2009, 8:http://www.microbialcellfactories.com/content/8/1/adry cell weight or glycerol [g/L]growthde-repression60 401b36 time [h]dry cell weight or glucose [g/L]100 start feeding 80 60 40 20 1 0 24 48 72 96 time [h] 120 144 0cdry cell weight or glycerol or methanol [g/L]growth de-repressioninduction 10060 4036 time [h]A, B, C: 3 Figure Schematic depiction of H. polymorpha-based fermentation processes PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27597769 A, B, C: Schematic depiction of H. polymorpha-based fermentation processes. The figures are modified versions of figures from previous publications. For further details see text. a fermentation of a hirudin-prod.
