1. Introduction
At the beginning of the 21st century, mankind faces the inevitable
depletion in global strategic petroleum reserves on earth, as
fossil fuels are not renewable. The increase in prices of petroleum
based fuels and environmental pollution issues have encouraged
research into the development of biofuel technology to produce
energy from sustainable resources [1]. Thus, it is urgent to look for
alternative energy sources and fuels that are sustainable and
environmentally safe [2e6]. Biodiesel, hydrogen and methane are
considered to be the energy of the longer term since they are
alternative energy sources with high energy content. Biodiesel is an
alternative fuel that has received a substantial attention in recent
years as a result of its degradable, non-toxic and low CO2 emission
[7]. The first generation of biodiesel productionwas depends up on
vegetables oil. By 2050 the world population should reached more than 9 billions of persons which resulted in increase energy demand
from 60 to 160%. This demand of biodiesel production will
compete with edible oil which causes increasing in food price.
Therefore, fungal lipids can represent a valuable alternative feedstock
for biodiesel production, and a potential solution for a biobased
economy [8]. Fungi that produce more than 20% (g g1)
biomass lipid are referred to as oleaginous microbes [9]. Filamentous
fungi hold promise as oil sources for biodiesel production as a
result of they can fairly rapidly (within 96e130 h) accumulate
biomass and substantial amounts of lipids, dominated by triglycerides.
Fungi of the order Mucorales fulfill all requirements for
being employed as core catalysts in biorefineries, and are considered
as potential producers of single cell oils [10], Cunninghamella
echinulata belongs to the order Mucorales within class Zygomycetes.
Mucoralean fungi are best known as saprophytes which favor
simple sugars as opposed to more complex molecules. Organisms
belonging to Mucorales have a growth strategy characterized by
short generation times, rapid absorption of sugars, and type of fatty
acid profile obtained. Hydrogen production through dark or photofermentative
conversion of organic substrates is of great interest due to its dual function of the major portion of municipal refuse
reduction and clean energy generation, thereby acting as a promising
option for biohydrogen production [11e13]. Anaerobic bacteria
capable of hydrogen production can use various sources of
renewable biomass and numerous agriculture, municipal and food
processing waste and wastewater sources [14e16]. Biogas as a sort
of biofuel, comprises primarily methane (CH4) and carbon dioxide
(CO2) and may have small amounts of hydrogen sulphide (H2S),
moisture and siloxanes. It may be used as a fuel in any country for
any heating purpose. Biogas could also be compressed, very similar
to natural gas. Biogas holds a good vary of applications, it may be
used as a replacement of fossil fuels in generation of power and
heat, and it also can be upgraded to vaporous vehicle fuel [17].
Biogas is formed by different groups of facultative and obligatory
anaerobic microorganisms. The main steps of the biogas production
are hydrolysis, acidogenesis, acetogenesis, and methanogenesis
[18e20].
The biosynthesis of lipid by oleaginous fungi was associated
with the formation of volatile fatty acids as metabolic products
[21]. These acids resulted in a sharp decrease in culture pH and
therefore inhibited the fungal growth [22]. But, it is somewhat
difficult to achieve the full utilization of molasses sugar to lipid by
fungal fermentation. This appears to be one amongst the most
important obstacles in themolasses fermentation process for lipid
biosynthesis. To avoid this drawback, the system outlined in the
present paper takes into consideration the non-utilizable sugar
and volatile fatty acids produced therein (spent medium).
Combining fermentative C. echinulata with strictly anaerobic
bacteria for hydrogen (Clostridium acetobutylicum ATCC 824) and
methane (methanogenic bacteria) production could provide an
integrated system for maximizing the biofuel yield from sugarcane
molasses. In such a system, the fermentation of spent medium
containing residual sugar and organic acids generated by
fungi, which are then converted into hydrogen by Clostridium in
the second stage. This stage mainly involves fermentative production
of hydrogen from residual sugars and formation of volatile
fatty acids and ethyl alcohol during acidogenesis and is
actually the precursor to methanogenic bacteria for methane
production in a complementary third stage.
The current study aimed to maximize the production of costcompetitive
biofuel from sugarcane molasses by medium recycling
through sequential three-stage fermentation. This system
adopted C echinulata as a potent lipid producer for generation of
biodiesel from sugarcane molasses and to investigate the influence
of some nutritio