Molecular Microbiology and Biotechnology Institute of Biology Leiden Leiden University, A.F.J. Ram
Treatment protocol
Strain and inoculum: Aspergillus niger N402 (cspA), a standard laboratory strain with short conidiophores (Bos et al., 1988), was propagated on solidified Complete Medium (CM) to produce conidial inoculum for submerged cultivation. CM agar plates contained (per liter):10.0 g glucose, 6.0 g NaNO3, 1.5 g KH2PO4, 0.5 g KCl, 0.5 g Mg SO4∙7H2O, 1.0 g casamino acids, 5.0 g yeast extract, 20 g agar and 1 ml trace metal solution. The trace metal solution contains per liter, 10 g EDTA, 4.4 g ZnSO4⋅7H2O, 1.01 g MnCl2⋅4H2O, 0.32 g CoCl2⋅6H2O, 0.315 g CuSO4⋅5H2O, 0.22 g (NH4)6Mo7O24⋅4H2O, 1.47 g CaCl2⋅2H2O and 1 g FeSO4⋅7H2O (modified from composition given by Vishniac and Santer, 1957). pH of the medium was adjusted to 5.8 with NaOH prior to autoclaving. The solid medium cultures were incubated for at least 6 days at 30 °C to allow adequate conidium formation. Spore plates were stored for no more than three months at 4 °C. For preparation of inoculum, conidia were harvested and washed with a sterile detergent solution containing 0.05 % (w/v) Tween 80 and 0.9 % (w/v) NaCl. Batch cultures: Bioreactors holding 5 liter (kg) Minimal Medium (MM) was inoculated with conidial suspension to give 109 conidia L-1. The MM contained (per liter): 4.5 g NH4Cl, 1.5 g KH2PO4, 0.5 KCl, 0.5 MgSO4⋅7H2O and 1 ml trace metal solution (described above). pH was adjusted to 3. The final-cell-density-limiting (growth-limiting) substrate was 4 g maltose (monohydrate) per liter. Concentrated solutions (20x) of carbon source were heat sterilized separately and added aseptically to sterile MM. During cultivation, temperature was 30 °C and pH 3, kept constant by computer controlled addition of 2 M NaOH or 1 M HCl. Sterile air was supplied at 1 L min-1 through a ring-sparger. Dissolved oxygen tension was above 40% of air saturation at anytime, ensuring oxygen sufficient growth. Germination of the dormant conidia was induced by addition 0.003% (w/w) yeast extract before inoculation. During the first six hours of cultivation, the culture was aerated through the headspace of the reactor and stirrer speed was kept low at 250 rpm. These precautions minimized gas-liquid interface, thereby reducing loss of hydrophobic conidia to the exhaust gas. After six hours and germination of most conidia (now hydrophilic), air was sparged into the culture broth and mixing intensified (750 rpm) for more efficient mass transfer. At this stage, 0.01% (v/v) PPG was added as antifoam agent. Submerged cultivation was performed with 6.6 L BioFlo3000 bioreactors (New Brunswick Scientific, NJ, USA). Culture mass was monitored with a Mettler Toledo KCC150s scale, which has a maximum load capacity of 150 kg and an accuracy of 10g. The glass surface of the head space was cooled to minimize wall growth. Cell retention: A special cell retention device (FIG. 1A) was developed to enable efficient separation of filamentous biomass and allow reliable operation of retentostat cultures (this study). The retention principle is simple (FIG. 1B,C). Dilute culture broth rises, by unforced filtration over a coarse filter, into a cylinder inserted from the top-plate (through existing 19mm injection port). The dilute is constantly drained from the cylinder, thereby maintaining constant culture volume. The vertical position of the filter can be adjusted aseptically to facilitate filtration as biomass density increases. An anti-fouling mechanism prevents blockage of the filter by intermittent sparging (2s every 12s) sterile air (2 L min-1) through the cylinder and filter. Biomass retention was nearly complete (95-99%) and filtrate could be drained at a rate of at least 0.25 L h-1 (0.19L h-1 per cm2 filter area) at biomass densities of at least 10 g dry weight per liter culture broth. The device was constructed from durable materials with high temperature resistance, this allowed repeated use and heat sterilization. The filter housing (frame and cylinder, 19x200mm) was made of stainless steel, the septum of silicone and drain tubing of Teflon. The filter itself was cut from sheets of Spectra Mesh (Spectrum Laboratories, CA, USA). Stainless steel mesh with pore size of 51 µm and 42% open area was used. The cell retention device was crafted at the BMB work shop (University of Southern Denmark) according to instructions from T. R. J. Biomass sampling: Biomass for total RNA extraction was sampled at day 2 after start of retentostat flow.
Growth protocol
see Treatment Protocol
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from homogenized mycelial samples using TRIzol reagent (Invitrogen).
Label
Biotin
Label protocol
The Affymetrix One-Cycle Target Labeling Kit and Control Reagents (#900493) are used to synthesize Biotin-labeled cRNA. From each RNA sample 2 μg is used for the labeling experiments.
Hybridization protocol
The GeneChip Hybridization, Wash and Stain Kit (#900720) is used for the for the hybridizations, washing, staining and scanning of the chips. The Affymetrixprotocols are strictly followed. The Affymetrix custom Aspergilus niger Genome Arrays are used for hybridization. To the 270 μl hybridization cocktail, 30 μl labeled material is added according the manual of the Affymetrix One Cycle Target Labeling Kit.
Scan protocol
Standard Affymetrix protocol
Description
n/a
Data processing
We used the Mas5 algorithm (as implemented in Genedata Expressionist Pro 4.5) for preprocessing of .CEL files. Genes with detection calls of present (p<0.04) or marginal (0.04<=p<0.06) in at least one of three replicate measurements were accepted as expressed and used in further analyses. Raw signal values were subjected to scaling normalization to the arithmetic mean of a reference group (Day 0 sample) prior to comparison.
