2.4. Solid state fermentation and production of nutrient
supplements
SSF with A. oryzae was carried out using the LF as the sole solid
substrate in 250mL Erlenmeyer flasks at 30 ◦C according to the
methodology described by Kachrimanidou et al. (2013). LF solids
(5 g) were sterilized at 121 ◦C for 20 min and were subsequently
inoculated with a fungal spore suspension (106 spores mL−1). The
initial moisture content was adjusted at 65% (w/w, on dry basis).
The pH value was not controlled during SSF.
After the end of SSF, fermented solids were macerated using a
kitchen blender after suspension in either 500mL of tap water or
500mL of the residual soluble fraction (Fig. 2). When tap water was
used, the suspension was mixed with unprocessed LF for the production
of hydrolysate I. When the residual soluble fraction was
used, the suspension was mixed with the SR fraction generated
after alkaline solubilization of proteins (Fig. 2) for the production
of hydrolysate II. Enzymatic hydrolysis was carried out using either
50gL−1 or 100 g L−1 of initial solid concentration. Hydrolysis was
performed in 1 L Duran bottles at 45 ◦C and uncontrolled pH value.
Agitation was conducted using magnetic stirrers. Remaining solids
were removed by vacuum filtration and all hydrolysates were subsequently
filter sterilized using a 0.2mfilter unit (Polycap TM AS,
Whatman Ltd.). The pH value of the produced hydrolysates was
adjusted to 6.7–6.9 for optimum C. necator growth with 5MNaOH.