Microorganisms and culture:
Aspergillus niger: The fungal culture Aspergillus niger was screened from different soil samples of local paddy
and groundnut fields and identified with the help of manuals like “Dermataceous fungi” by Barnett (20), “Text
book of Mycology” by Alexopolus (21) and “Handbook of soil fungi” by A. Nagamani, I K Kunwar and C.
Manoharachary (22). The fungus was cultured and maintained on Potato Dextrose agar medium at 300
C. After
optimum growth the culture was stored at 40
C in refrigerator for further use.
Preparation of Inoculum: For the preparation of inoculum the culture was plated on PD agar plates. The plates
were incubated at 300
C for 72 hrs until the mycelium sporulates black conidia. Inoculum was produced in 250
ml Erlenmeyer flasks containing 100 ml potato dextrose broth by transferring 2 discs from the PDA plates. The
flasks were incubated for another 72 hrs at 300
C till the mycelial mat develops. This mycelial mat was used as
inoculum in further saccharification experiments.
Saccharomyces cerevisiae: The yeast Saccharomyces cerevisiae was isolated from soil samples collected from
vineyards rich in waste materials which include fallen and discarded grapes. Samples were collected in sterile
containers and transferred to laboratory. The soil samples were suspended in sterile distilled water and allowed
to settle, then the supernatant was diluted by serial-10 fold dilutions and the samples were inoculated on to
sterile Yeast-extract, Peptone and Dextrose (YEPD) plates. The plates were incubated at 300
C for 48 hrs. The
grown yeast isolates were identified as Saccharomyces cerevisiae by studying some of the morphological,
biochemical and physiological characteristics (23).
Setting up of fermentation: The fermentative production of bioethanol was carried out in two steps- a)
saccharification and b) fermentation. Two methods i.e. stationary and shaking were adopted. The chemically
pre-treated substrates were used for all the experiments. In order to optimize bioethanol production the
substrates were taken in three different variations in the following manner (Table-1).