Green Fuel Technology. Microbial Oil Production from Oleaginous Yeast (Cryptococcus curvatus)
©2016
Textbook
47 Pages
Summary
The increased industrialization and motorization has led to a steep rise for the demand of petroleum products. Petroleum based fuels are obtained from limited reserves. It is estimated that the known crude oil reserves could be depleted in less than 50 years at the present rate of consumption. Hence, it is necessary to look for alternative fuels. An entire branch of biotechnology, referred to as "white biotechnology", centers on the bio production of fuels and chemicals from renewable sources. About 90% of the current biofuel market is represented by biodiesel and bioethanol. Biodiesel is an efficient, nontoxic, biodegradable and clean burning fuel alternative to petroleum fuels which runs in unmodified diesel engines. This paper reviews approaches to microbial synthesis of biodiesel, focusing on the role of synthetic biology as an enabling technology in the design of optimal cell factories.
Excerpt
Table Of Contents
LIST OF ABBREVATIONS
FAEE Fatty Acid Ethyl Ester
RSM Response Surface Method
TAG Triacylglycerides
STE
s
Steryl Esters
FAME
s
Fatty Acid Methyl Esters
FAAE
s
Fatty Acid Alkyl Esters
OR Operations Research
CPM Cost per thousand household exposures
to this Media Vehicle
FFA Free Fatty Acid
MTCC Microbial Tissue Culture Collection
AOAC Association of Official Analytical Chemists
KH
2
Po
4
Potassium dihydrogen phosphate
MgSO4.7H2O
Magnesium sulphate
NaCl Sodium Chloride
CaCO
3
Calcium Carbonate
KCl Potassium chloride
CO Carbon Monoxide
HC HydroCarbons
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ABSTRACT
The increased industrialization and motorization has led to a steep rise for
the demand of petroleum products. Petroleum based fuels are obtained from
limited reserves. Hence, it is necessary to look for alternative fuels, which
can be produced from oleaginous yeast
Cryptococcus curvatus. The
objective of the project is medium optimization for economical production
of Biomass and Microbial oil by the well known oleaginous yeast
Cryptococcus curvatus and conversion of the lipids to biodiesel (Fatty Acid
Ethyl Esters). The media was optimized using the design software
STATEASE. Variables with best optimized value for the high yield of
Biomass was screened by Plackett Burmann Method. Then the optimum
concentration of those screened variables was predicted using
RSM(Response Surface Method). The extraction method Folch
Extraction method was used for the extraction of microbial oil .
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1. INTRODUCTION
In recent times, The world has been confronted with an energy crisis due to
depletion of resources and increased industrialization, motorization and increased
environmental problems. on the other hand with population increasing rapidly and
many developing countries expanding their industrial base and output, worldwide
energy demand is bound to increase. It is estimated that the known crude oil
reserves could be depleted in less than 50 years at the present rate of consumption.
Biodiesel is an efficient, non toxic, biodegradable and clean burning fuel
alternative to petroleum fuels which runs in unmodified diesel engine. Diesel fuel
is largely utilized in the transport, agricultural, commercial, domestic, and
industrial sectors for the generation of power / mechanical energy, and the
substitution of even a small fraction of total consumption by alternative fuels will
have a significant impact on the economy and the environment
Global warming and the continued depletion of non-renewable fuel resources are
two major problems that entangle our planet today and demand immediate
solutions . The extensive use of fossil fuels has caused greenhouse gas emissions
and damage to the environment, and has also led to the current instability of oil
supplies and continuous fluctuations in prices. These factors, which revolve around
economic, environmental and geopolitical issues, are central to the continued
interest seen in renewable energy sources . An entire branch of biotechnology,
referred to as "white biotechnology" , centers on the bioproduction of fuels and
chemicals from renewable sources. For biofuels, delicate optimization, and fine
tuning of these processes to maximize productivity and yield is of particular
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concern, as the viability of any biofuel process is extremely sensitive to factors
related to both raw material supply and production costs.
About 90% of the current biofuel market is represented by biodiesel and
bioethanol. However, bioethanol is not seen as an ideal biofuel for the future
because of its low energy density and incompatibility with the existing fuel
infrastructure . On the contrary, biodiesel is already better established and is
preferable to petrodiesel in terms of several characteristics, such as environmental
friendliness, renewability, reduced emissions, higher combustion efficiency,
improved lubricity, and higher levels of safety . Chemically, biodiesel comprises a
mixture of Fatty Acid Alkyl Esters (FAAEs). The most commonly used method to
produce biodiesel is the in vitro transesterification process, where
Triacylglycerides (TAGs) of vegetable oils are combined with methanol to form
Fatty Acid Methyl Esters (FAMEs) and the byproduct glycerol Alkalies (e.g.,
sodium hydroxide, potassium hydroxide, sodium metoxide, and potassium
metoxide) , acids (e.g., sulfuric acid) , or enzymes can be used to catalyze this
reaction . However, issues related to high cost and limited availability of vegetable
oils have become growing concerns for large-scale commercial viability of
biodiesel production . Also, the in vitro transesterification reaction presents some
unresolved issues, such as the need to use large amounts of toxic compounds
(sodium hydroxide, sulfuric acid, or methanol) and the high cost of isolation and
immobilization of enzyme catalysts . Various approaches to addressing these
problems have been explored. First, increasing interest in developing microbial
processes for the production of biodiesel from a wide range of other raw materials
may represent a promising alternative to the vegetable oils. Second, technologies
now exist that use living cells to synthesize products that are more easily
biodegradable, require less energy, and create less waste during production than
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those obtained by chemical synthesis. In order for a fermentation process to
compete with existing petroleum-based processes, the target molecule must be
produced at high levels of yield, titer, and productivity. These goals can be difficult
to attain with naturally occurring microbes. While metabolic engineering has
enabled extraordinary advances in the redesign of pathways for efficient target
molecule production, including biofuels , tools from synthetic biology make it
possible to create new biological functions that do not exist in nature. Essentially,
this is achieved either by heterologous expression of natural pathways or design of
de novo pathways. This paper reviews approaches to microbial synthesis of
biodiesel, focusing on the role of synthetic biology as an enabling technology in
the design of optimal cell factories.
1.1 Biofuel feedstocks
Because of its abundance and renewable nature, biomass has the potential to
produce extensive supplies of reliable, affordable, and environmentally sound
biofuels to replace fossil fuels. Many biomass feedstocks, which include
lignocellulosic agricultural residues as well as edible and nonedible crops, can be
used for the production of biofuels.
More than 95% of global biodiesel production now begins from virgin edible
vegetable oils which account for about 80% of the total production cost. However,
the socioeconomic impacts of large-scale biodiesel production from edible
feedstocks can be significantly lowered by the use of alternative feedstocks such as
nonedible oils or lignocellulosic biomass. The use of nonedible vegetable oils is
especially significant for biodiesel production in developing countries ,because of
the tremendous demand for edible oils as food. Increasing attention is also now
being given to the use of microbial oils as biodiesel feedstock, which are produced
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by certain oleaginous microorganisms .Lignocellulosic biomass, on the other hand,
is the largest known renewable source of carbohydrates. It generally consists of
about 25% lignin and 75% carbohydrate polymers (cellulose and hemicellulose)
.These polymers, upon complete hydrolysis, yield a mixture of hexose (glucose,
galactose, and mannose) and pentose (arabinose and xylose) .Synthetic biodiesel
can be produced from this renewable carbon source using the Fischer-Tropsch
process. Although conversion of lignocellulose into biofuels appears simple in
theory, the techniques used in this field are not fully established. The main reason
for this lag is the recalcitrant nature of cellulose and the toxic nature of the
products of lignin degradation. Several prokaryotes and eukaryotes that have
cellulolytic properties and are tolerant to the toxic products of lignin degradation
have been identified . However, the yield and productivity of biofuels synthesized
in this way are not sufficient to meet current energy demands. An efficient
cellulolytic organism should be able to hydrolyze lignocellulose completely,
ferment all sugars of lignocellulosic hydrolysate simultaneously, and tolerate toxic
compounds of lignin without compromising productivity .Therefore, for cost-
effective production of biofuels, the fuel-producing hosts must be designed.
In terms of ethanol production, starches (maize, wheat, barley, etc.) and sugar-rich
biomass (grasses, maize leaves, beets, sugar cane, etc.) have been the feedstocks
most commonly used for their bioconversion .However, advances in metabolic
engineering and synthetic biology have provided new tools for creating desirable
phenotypes for the production of ethanol from lignocellulosic biomass .Since
ethanol is one of the substrates used for in vivo synthesis of biodiesel, advances in
terms of maximization of two-carbon alcohol production from the most
economically viable feedstocks will be discussed later in this review.
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1.2 Synthetic Biology and Biodiesel Production
Synthetic biology emerged around the year 2000 as a new biological discipline,
and many different definitions have been applied to this field. However, one
commonly used way to describe synthetic biology is as the design and construction
of new biological functions that are not found in nature. Synthetic biology is a
discipline encompassing contributions from many fields , but this review places
particular emphasis on the design of microbes, either by modification of existing
pathways or by heterologous expression of natural pathways, in order to allow
efficient production of biodiesel. In this connection, the synthesis of biodiesel
using microbes is currently a highly promising alternative to conventional
technologies. Microbial biodiesel production has been approached from two
different angles: (1) by indirect synthesis from microbial oils, which are produced
and harvested for use in the conventional in vitro transesterification process, and
(2) by direct biodiesel synthesis using redesigned cell factories to increase
production of alcohols and/or FFAs, which are subsequently used for in vivo
synthesis of biodiesel. In the following sections, both approaches are reviewed.
1.3 Indirect Synthesis of Biodiesel from Microbial Oils
It is well known that many microbes, including certain types of microalgae,
bacteria, filamentous fungi, and yeasts, can accumulate intracellular lipids,
primarily TAGs, with these representing a large proportion of their biomass . Oils
derived from these oleaginous microbes represent promising raw materials for
biodiesel production through transesterification using the plant-based process.
The use of microbial oils offers several advantages when compared to plant-
derived oils . However, oleaginous microbes have varying prospects in the
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biodiesel industry. For example, microalgae are photoautotrophic microorganisms
that can convert CO
2
directly to lipids, which can then be used for biofuel
production, particularly for biodiesel . The oil content of microalgae usually ranges
between 15 and 70% by weight of the dry biomass . Scaling-up process for
autotrophic microalgae is complex, however, since light is needed during
cultivation. Although it is known that algae could be grown in dedicated artificial
ponds for generating biodiesel , the harvesting of miles and miles of algae growth
is required in order to generate substantial amounts of biodiesel. Thus, while the
microbiological aspects of this approach are extremely promising, the engineering
aspects pose the greatest challenge. Tools from synthetic biology have been
effectively used to convert certain autotrophic microalgae into heterotrophic
microorganisms . Essentially, this consists of the introduction of nonnatural
metabolic pathways into the autotrophic microalgae, thereby, allowing cultivation
using an organic carbon source instead of photosynthesis from sunlight.
1.4 Direct Synthesis of Biodiesel from Microbial Oils
Methanol, conventionally used as part of in vitro transesterification, is largely
derived from nonrenewable natural gas and is also both toxic and hazardous. On
the contrary, ethanol can be naturally produced from renewable resources, while
exhibiting low levels of toxicity and a higher degree of biodegradability. Ethanol
produced endogenously can therefore be used for in vivo synthesis of Fatty Acid
Ethyl Esters (FAEEs) with exogenously added FFAs. Similarly, microbial FFAs
can be used as feedstock for in vivo production of biodiesel, instead of TAGs from
vegetable oil.
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2. REVIEW OF LITERATURE
2.1
Medium optimization for the Production of Microbial oil
2.1.1 Medium optimization
The idea of optimization derives from an engineering discipline called "Operations
Research" and known as "OR". OR consists of a set of tools and approaches
known respectively as "algorithms" and "heuristics". Algorithms are mathematical
equations, while heuristics are fuzzier methods i.e. they are not equations. Both
algorithms and heuristics are aimed at improvement in the operations of an
organization. "Optimization models" are a type of algorithm intended to provide
the best possible solution to some problem facing an organization. Where the
problem itself is so complex that finding the best possible solution could cost more
than the benefit of doing so, the optimization models generally do not attempt to
find the best possible solution, but instead seek to find extremely good solutions
within reasonable cost and time parameters. This in fact is the more common
situation. Although in the latter case what is sought is literally "improvement"
rather than "optimization", these models are still conventionally called
optimization models in all cases. Within the guts of the model, all information
about a particular media vehicle are generally reduced to a single number
representing its cost: benefit value. A simplified example of such a cost:benefit
value is the CPM (Cost Per Thousand household exposures to this media vehicle).
A slightly less simplified example is the CPM Targets (Cost Per Thousand Target
Audience exposures to this media vehicle). The model is designed to maximize
value by selecting those vehicles with the lowest cost in relation to whatever
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parameter is to be maximized. The parameter to be maximized (e.g. Target
Audience exposures) is technically known as the "objective function". The builders
of media optimization models studied the way media planners and buyers
conventionally selected media vehicles, and constructed their models to mirror
these conventional procedures. In doing so, they sought to move from the
heuristics being used by planners/buyers into the use of true algorithms instead.
2.2 Raw material for the Production of Microbial oil
2.2.1 Yeast as the source for Production
The efficiency of oil biosynthesis by yeast and its composition depends on the
genetic properties of the yeast strains, cultivation conditions and the composition
of culture medium. Lipids are important storage compounds in yeast. Storage lipids
are usually found within special organelles knows as lipid particles or lipid bodies.
In yeast, these lipid bodies accumulate during stationary phase and they can
constitute up to 70% of the total lipid content of the cell.
Triacylglycerols (TAGs) and Steryl Esters (STEs) are the most important storage
lipids of eukaryotes cells such like yeast cells. TAG provides an energy source on
one hand and a source of fatty acids for membrane phospholipid formation on the
other hand. Mobilization of STE sets sterols free, which are also required for
membrane proliferation, especially of the plasma membrane. In the yeast as in
other eukaryotic cells, TAG and STE form the core of the so-called lipid particles
which are surrounded by a phospholipids monolayer with a small amount of
proteins embedded.
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Details
- Pages
- Type of Edition
- Erstausgabe
- Year
- 2016
- ISBN (PDF)
- 9783960675440
- ISBN (Softcover)
- 9783960670445
- File size
- 6.3 MB
- Language
- English
- Institution / College
- Arunai Engineering College – Department of Biotechnology
- Publication date
- 2016 (May)
- Keywords
- Biotechnology Bioengineering Biological engineering Petroleum product Petroleum based fuel biodiesel White biotechnology
