1. IntroductionBecause of diminishi

1. Introduction
Because of diminishing crude oil resources and increasing fuel
costs in recent years, ethanol has re-emerged as an alternative to
petrochemical-based liquid fuels. Ethanol is a renewable and clean
fuel produced using biomass as the raw material. It is by far the
most widely used biofuel for transportation worldwide [1]. In
Thailand, gasohols E10 and E20 (with mixtures of 10% and 20% ethanol,
respectively, in gasoline) have been widely used in vehicles,
and there are attempts underway to promote the use of E85 in
vehicles in the near future. According to the report on net renewable
energy analysis in Thailand, a positive net energy of 5.95 MJ/L
was obtained from the molasses-based ethanol system [2].
Traditional ethanol industries produce ethanol by batch or fed-batch
processes. Cell immobilization in continuous fermentation
has been proposed as an effective means of improving the ethanol
production rate. Cell immobilization could lead to a higher biomass
concentration and enhanced biological stability. The protection of
immobilized cells against toxicity has been reported previously
[3,4]. Almost all cell immobilization methods are based on adsorption
or entrapment. Gel entrapment methods usually involve the
problems of gel degradation and limitations of oxygen, nutrient
and metabolite mass transfer. In contrast, natural adsorption is generally
a simple and inexpensive procedure for cell immobilization
without internal mass transfer limitations. Moreover, at the end
of the fermentation, the carriers of yeast cells adsorbed onto
agricultural byproducts can be modified into an animal feed co-product.
Various bio-materials have been examined as support
materials for yeast cell immobilization, including wood blocks [5],
porous cellulose [6,7], apple cuttings [8], loo-fa sponges [9,10], and
sorghum bagasse [3].
In this work we attempt to develop a new and effective method
of yeast immobilization for ethanol production. A thin-shell silk cocoon
(TSC), a residual from the silk industry, was used as a natural
support material because of its advantages, which include its biocompatibility,
light weight, high strength, low cost, chemical stability,
high porosity and high surface area. TSC is the shell cocoon
after removing the outer silk layer. It has been reported to have
special microstructure, good mechanical strength when wet, resistance
to enzymatic cleavage and high oxygen permeability [11].
Silk filaments adhere to form the cocoon shell. The filament is
composed of two proteins: fibroin and sericin. Fibroin has a high
content of the amino acids glycine and alanine. Sericin is characterized
by its high content of serine and 18 amino acids, including
essential amino acids [12]. There have been reports on the improved
activity and stability of immobilized enzymes using silk fibroin
as a support [13].
Raw silk is produced in many regions throughout Thailand.
However, the silkworms are grown primarily in the northeast region.
According to the report by EU-Thailand Small Projects Facility
in 2007, the world production of raw silk was about 135 thousand
tons in 2004. Thailand was the sixth largest raw silk producing
country with the production about 1550 tons per year. In Thailand,
a few amounts of TSCs are used as handicraft making raw material,
as material for filling pillowcase or as scrub cocoons for traditional
remedy. The leftovers are used as fish food and fertilizer. Owing to
its substantial quantity, low price and advantageous properties, it
has the potential to be used as immobilizing material for ethanol
industries. Therefore, in this study, thin-shell silk cocoons were
used for the natural immobilization of yeast cells for ethanol
production in repeated batch and continuous fermentations of
blackstrap molasses. To the best of our knowledge, this is the first
report of the use of silk cocoons for yeast cell immobilization.