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New Inuliolytic Fungus: Molecular Identification and Statistical
Optimization of Its Inulinase Secretion for Bioethanol Production
from Helianthus Tuberosus
Mohammad Magdy El-Metwally1
, WesamEldin Ismail Ali Saber2 and Samia Abd Allah AbdAl-
1Department of Botany and microbiology, Faculty of Science, Damanhour University, Egypt
2Microbial Activity Unit, Department of Microbiology, Soils and Water and Environment Research
Institute, Agricultural Research Center, Giza, Egypt. 3Department of Nucleic Acids Research, Genetic Engineering and Biotechnology Research Institute, City for
Scientific Research and Technology Applications, Alexandria, Egypt.
Abstract. New inulinolytic fungus was identified as Talaromyces purpureogenus KJ584844, and investigated for maximum hydrolysis of inulin of Helianthus tuberosus tubers. NH4Cl and yeast extract were
found to be significant in inulinase production based on Plackett-Burman matrix, which increased the
inulinase production by 3.59-fold. The interaction between both variables was optimized based on central
composite design, with 2.35 fold increase in the enzyme activity (166.13 U g-1
tubers) after only 3 d of
incubation. The sugary material resulted (0.823 g g-1
tubers) from the hydrolytic action of the inulinolytic
fungus was subjected to bioethanol production by Saccharomyces cerevisae. A total ethanol yield of 0.367 g
per g tubers was recovered. This fungus is not previously reported as inulinolytic fungus, although its high
efficiency, additional optimization studies is needed for over secretion of inulinase to maximize the
hydrolytic efficiency of tubers into sugary materials, which is the building block for bioethanol production.
Keywords: talaromyces purpureogenus, inulinase; molecular identification; central composite design;
Biomass is a hidden form of energy and can be used instead of fossil fuels but the true challenge based
on the way by which we increase the impact of biomass utilization and conversion to available form of
energy source. In this article the selection of H. tuberosus based on its high tolerance as it can grow under
annual precipitation ranging from 31 to 282 cm, with suitable average temperature range of 6.3 - 26.6C, and
pH of 4.5 to 8.2 with no or minimal fertilizer requirement .
Like sugar beet, H. tuberosus produces sugars in the above ground and stores them in the roots and
tubers. The tubers consist of 7579% water, 23% proteins, and 1516% carbohydrates, of which inulin
constitute 80% or more. Inulin is a polyfructan consists of linear chains of -2, 1-linked D-fructofuranose
molecules terminated by a glucose residue through a sucrose-type linkage at the reducing end . Such
inulin source has recently received attention as a renewable raw material for fructose syrup production and
ethanol biosynthesis as well as acetone and butanol . It's too hardly and costly to do that without inulinase,
which targeting the hydrolases of -2, 1 linkage of inulin cleaving it into fructose and glucose.
Depending on their mode of action, microbial inulinases are classified into, 1) endoinulinases (2, 1- -
D-fructan fructanohydrolase; EC 126.96.36.199), which breaking bonds between fructose units inside the inulin
Corresponding author. Tel.: +2 01003956536 E-mail address: firstname.lastname@example.org
International Proceedings of Chemical, Biological and Environmental Engineering, Vol. 99 (2016)
DOI: 10.7763/IPCBEE. 2016. V99. 2
polymer to produce oligosaccharides, and 2) exo-inulinases (-D-fructohydrolase; EC 3.2.1 .80), which
producesingle fructose units from the non-reducing end of the inulin molecule -.
Fungi are the best source for commercial production of inulinases because of their easy cultivation and
high yields of the enzyme especially in solid state fermentation (SSF). SSF has many preferences to
submerged fermentation (SmF) for microbial enzyme production including: superior productivities, lower
operating costs, less demands for contamination control, cheaper fermentation media, good and higher
oxygen supplementation, simpler equipment and control systems and lower energy consumption.
The statistical and mathematical approaches have several advantages in the microbial enzymes
production, of which the design of Plackett-Burman introduces efficient method for screening and selection
among large numbers of tested variables without testing their interaction, the interrelationship among the
effective variables could be determined latter using response surface methodology (RSM). Central composite
design (CCD) is one of the popular designs of RSM used for such purpose. It provides statistical modeling
for understanding the interactions among the process parameters at varying levels and in calculating the
optimal level of each parameter for maximization of a given target .
Most studies on inulinase production utilize yeasts as the microbial model, on the other hand
optimization of SSF medium for the production of inulinase by fungi on tubers of H. tuberosus are limited,
and its application in bioethanol production are also rare. In this article, we expanded our knowledge by
adding a new molecularly identified inulinase-producing fungus. The SSF technique and the statistical CCD
were used to maximize the productivity of inulinase on tubers of H. tuberosus, the hydrolysate resulted from
the catalytic action of inulinase was applied in the bioethanol production.
2. Materials and Methods
2.1. Tubers of Helianthus Tuberosus Healthy clean tubers of H. tuberosus were obtained from the Horticulture research station, Agricultural
Research Center, Egypt (+7m altitude, 30 11" latitude and 28 26" longitude), during the summer growing
seasons of 2015. The tubers were cool-dried and grinded; the resulted powder was used as a solid-state for
the fermentation process.
2.2. Fungal Isolate The fungus was isolated previously from deteriorated textile sample and showed reasonable inulinase
activity among other isolates in primary screening test on inulin agar plates. The fungus was preserved at
4 C on slants of Czapek agar medium after incubated at 282 C for 7 days, and sub-cultured monthly.
2.3. Molecular Identification of the Fungus Isolation, amplification and molecular sequencing of Inter Transcribed Spacer (ITS) of the fungal DNA
was carried out. The genomic DNA of the fungus was extracted according to the procedure of Lee and
Taylor (1990)  with some modifications as follows; after 10 days of culture growth on PDA medium, the
mycelia were collected and frozen with liquid nitrogen, then grounded with sterilized mortar-pestle and kept
in 1.5 ml micro tube, to which equal amount of extraction buffer (50 mMTris-HCl, pH 7.5, 50 mM EDTA,
pH 8 and 1% sarkosyl) was added and incubated at 65 C for 30 min. After incubation, same amount of PCI
(25 ml phenol: 24 ml chloroform: 1 ml isoamyl-alcohol) was added, vortexed and centrifuged at 4 C, 10
min, 12000 rpm. Only the supernatant of upper part was taken in 1.5 ml micro tube, to which 1000 l of 99.9%
alcohol was added and centrifuged at 4 C, 5 min, 12000 rpm. In this case, the supernatant was removed, and
added 500 l of 70% alcohol with precipitated DNA, vortexed and centrifuged at 4 C, 5 min, 12000 rpm.
Again supernatant was removed and waited until residual alcohol evaporated. Finally, 500 l of sterilized
distilled water was added. DNA concentration was measured using spectrophotometer .
The ITS region of the rDNA of the isolated fungus was amplified by polymerase chain reaction (PCR)
using universal primers ITS1 (5'-TCCGTAGGTGAACCTGCG-3') and ITS4 (5'-
TCCTCCGCTTATTGATATGC-3') according to (White et al., 1990) . Amplification reaction was
performed in a total volume of 20 l containing 10 PCR buffer 2 l, dNTP 1.6 l, 0.5 l of each primer, 0.2
l ofTaq polymerase, 1l of genomic DNA and 14.2 l of sterilized distilled water. PCR reaction was
performed using thermal cycler (Eppendorf Thermal Cycler) with an initial denaturation stage of 5 minutes
at 95 C, followed by 35 cycles of denaturation for 30 seconds at 94 C, annealing for 30 seconds at 52 C,
extension for 1 minute at 72 C and a final extension for 10 minutes at 72 C. Amplification products were
electrophoresed on 1.5% agarose gel with a 1,3 kb DNA ladder as marker and purified using PCR
purification kit FERMENTAS K 0701 and finally sequenced using the ABI PRISM dye cycle sequencing
ready kit (PerkinElmer) and an ABI PRISM377 sequencer according to the manufacturers protocol (USA)
using the forward ITS primer. DNA sequencing and chain terminating inhibitors was achieved as described
by Sanger et al. (1977) . ITS sequence was submitted, deposited and aligned into GenBank to obtain
similarities of the target sequence and the closely related fungi sequences.
The evolutionary distances were computed using the Maximum Composite Likelihood method  and
are in the units of the number of base substitutions per site. All positions containing gaps and missing data
were eliminated from the dataset (Complete deletion option). Phylogenetic analyses were conducted in
2.4. Setting up Plackett-Burman Matrix The