Sustainable Chemistry and Pharmacy 6 (2017) 82–89

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Sustainable Chemistry and Pharmacy

journal homepage: www.elsevier.com/locate/scp

Perspectives for the use of biotechnology in green chemistry applied to

T
biopolymers, fuels and organic synthesis: from concepts to a critical point of
view
Maíra Fasciotti
Instituto Nacional de Metrologia, Qualidade e Tecnologia – INMETRO, Divisão de Metrologia Química e Térmica, Diretoria de Metrologia Científica e Industrial, 25250-
020, Xerém, Duque de Caxias, Brazil

ARTICLE INFO ABSTRACT

Keywords: Since the eighteenth century, after the first industrial revolution, humans have been exploiting the planet's natural resources in
Biotechnology an unsustainable manner. This has caused some irreversible impacts on the environment. Because of this, we are facing a
Green chemistry change of philosophy within the scientific community about chemical and industrial processes. During the 1990s, the concepts
Biofuels of green chemistry began to solidify, while in parallel some major advances in biotechnology were achieved through
Biopolymers
developments of genomic sciences and genetic and metabolic engineering. This work will discuss some new insights into the
Organic synthesis
use of biotechnology as an important tool in green chemistry, showing new applications to biopolymers, biofuels and as a new
alternative to tradi-tional organic synthesis, making chemical processes more sustainable and less damaging to the planet.

1. Background
Since the mid-eighteenth century with the first industrial revolu-tion,
mankind has been changing the way of extracting resources from the planet.
Initially, we implemented a basically artisanal production system, in which
humans themselves were the main production tools (de Vries, 1994). This was
drastically altered by the introduction of machinery, driven by energy derived
from fossil fuels such as coal (Fahrenkamp-Uppenbrink, 2015; Berg, 1978),
mainly in the textile and metallurgical industries, which as a result expanded
dramatically (Fores, 1981). At the end of the nineteenth century, the world
experi-enced the second industrial revolution, in which rapid technological
development led to most of the industrial advances that allowed the sale of
consumer goods that still are important to the society today. This brought the
establishment of the automotive and aviation industries, production of oil-
derived materials, the pharmaceutical industry, and computing (the base for
the ongoing information revolution) (Spilhaus, 1970).
2009). After all, as proposed by the classic thought of A.L Lavoisier:
"Nothing is lost, nothing is created, everything is transformed." There-fore,
the industrial waste will continue to "exist" on this planet as long as a better
destination is not given to it, because the natural processes of degradation are
much slower than that of the producing new waste. Moreover, most of these
advances depend on energy derived from fossil fuels, which besides
contributing to global warming and other en-vironmental problems are not
renewable (at least not in the "short" run).
As known, pollution has caused much irreversible damage to the
environment. The increase in the anthropogenic emission of greenhouse gases
(GHG)1 from burning fossil fuels and forest destruction is now a global
concern that demands a unified effort from all nations, with the aim of slowing
global climate change (Allison and Bassett, 2015). The rapid industrialization
processes has also caused damage in other spheres, such as soils and water,
impacting in multiple ways the life quality of all living beings (Kelly and
Fussell, 2015; Grossman and Krueger, 1995).

However, the unsustainable exploitation of Earth's natural resources in the
last 300 years has obviously caused impacts that threaten the well-being of
future generations. The waste generated as a consequence of all the industrial
advances of recent centuries is not adequately treated or sufficiently recycled,
posing one of the greatest challenges to society (Giusti, 2009; El-Fadel et al.,
1997; Houk, 1992; Robinson,
In the twenty-first century, although belatedly, society in general and the
scientific-industrial community now largely agree that the ways of interacting
with the environment and exploiting resources need to be changed. In the
midst of the third industrial revolution, characterized by an extremely refined
and advanced chemical industry, we are also facing a new revolution that
comes from the incredible advances of the

E-mail address: mfasciotti@inmetro.gov.br.

1 Green house gases abrangem CO2, N2O, CH4 e gases fluorados.

http://dx.doi.org/10.1016/j.scp.2017.09.002
Received 23 March 2017; Received in revised form 27 September 2017; Accepted 28 September 2017
Available online 13 October 2017
2352-5541/ © 2017 Elsevier B.V. All rights reserved.
M. Fasciotti
genomic sciences (Abelson, 1998). The general hope is that this current
revolution can address the impacts generated by the previous un-sustainable
revolutions.
2. Important concepts: green chemistry, biotechnology and white
biotechnology
With the change in the mentality towards the planet's serious en-
vironmental problems and the general acceptance of the fact that something
needed to be done to mitigate the damage, the concept of "green chemistry"
was developed, which basically encourages a closer look at the impacts of
industrial processes on nature.
The first principle of the United Nations Conference on Environment and
Development, which met in Rio de Janeiro from 3 to 14 June 1992, states
that:
“Human beings are at the center of concerns for sustainable development.
They are entitled to a healthy and productive life in harmony with nature.”
The role of chemistry to achieve this goal is undeniable. According to
data from the United States Environmental Protection Agency (US-EPA),
around 278 million tons of toxic wastes were generated in 1991, of which the
greatest portion came from the chemicals industry (Sanders, 2011). As a
result, the biggest corporations, such as Dow Chemical and DuPont, have
begun to devote efforts to comply with increasingly restrictive environmental
regulations, thereby tapping into green chemistry practices. This term, which
was proposed by the che-mist Paul Anastas in the 1990s, basically aims to
develop and imple-ment chemical products and processes to reduce or
eliminate both the use and generation of substances harmful to human health
and the environment, by applying the twelve basic principles of green chem-
istry, already well known (Anastas, 1998).
In practice, these concepts of green chemistry have revolutionized the
way of planning new projects and research in chemistry and have impacted its
sub-areas in the most diverse applications (Horváth and Anastas, 2007;
Fantke et al., 2015). Moreover, in parallel with this change of mentality, there
have been significant advances in bio-technology, which - under some
circumstances – can provide valuable or alternative tools for the
implementation of some of the 12 concepts of green chemistry.
Therefore, biotechnology is a general term used over a very broad field of
study. According to the Convention on Biological Diversity, biotechnology
means (Zaid et al., 1999):
"Any technological application that uses biological systems, living or-
ganisms, or derivatives thereof, to make or modify products or processes
for specific use."
It is an interdisciplinary area of science in which the main focus is
studying the use of cells, microorganisms, in vitro or in vivo, for the
production of new products through new processes. It is also an area of
science that has been applied for centuries: a simple fermentative process for
the production of alcoholic beverages can be considered a biotechnological
process, since it uses microorganisms to transform sugar into alcohol.
However, the main breakthrough in biotechnology was the provided by
significant advances in the genomic sciences (Cañestro et al., 2007), and of
genetic and metabolic engineering (Parekh, 2004).
The genomic sciences have had a great impact on the chemical in-dustry,
since it is now possible to genetically modify microorganisms to target certain
metabolic behavior according to what one wishes to obtain. This is called
metabolic engineering and these genetically modified microorganisms are
known as "engineered" microorganisms (Parekh, 2004). Thus, instead of
thinking about a longstanding che-mical process that has reagents, catalysts,
products and byproducts, the chemical process is designed to use some kind
of starting material, such
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Sustainable Chemistry and Pharmacy 6 (2017) 82–89
as an industrial waste, and some microorganism or enzyme capable of
transforming it into the desired final material or product. In practice, this
process offers advantages to traditional methods, by improving the selectivity
of chemical processes. In addition, genetic engineering offers new pathways
to obtain desired products, sometimes with lower pro-duction costs and using
safer and more sustainable materials.
During recent decades, biotechnology has been applied in several areas of
medicine, for the development of new pharmaceutical drugs (Olsen et al.,
2004; Drews and Ryser, 1997; Orive et al., 2003; Butcher et al., 2004),
vaccines and antibodies (William, 2005; Luckow and Summers, 1988), and
medical materials for transplants or prostheses (Yang et al., 2014; Gaharwar
et al., 2014; Ding et al., 2014; Fu et al., 2013; Hench and Polak, 2002); in the
area of food and agriculture, to produce eco-friendly food (Andrés et al.,
2016; Alam et al., 2014; Kasmi et al., 2017); and for environmental
protection, with the development of new biofuels derived from plants and
other renewable sources (Peralta-Yahya et al., 2012; Georgianna and
Mayfield, 2012), among other applications. In the early 2000s, more than 120
products obtained through biotechnology processes had been approved by the
FDA (U.S. Food and Drug Administration) (https://www.bio.org/media/press-
re-lease/biotechnology-fundamentals-solid, 2016).
In this way, biotechnology is a science that can be applied as a tool to
improve processes in virtually unlimited applications. It may be the key
science by making traditional chemical processes into "green" processes,
where the combination of biotechnology and green chem-istry leads to so
called "white biotechnology". It is an area that has emerged rapidly in recent
decades and refers to the use of biotechno-logical approaches in the
laboratory and industrial production of fine chemicals, biofuels and
agricultural products, among many others (Gupta and Raghava, 2007). The
main focus of white biotechnology is the development of "clean" processes
that result in reduction of green-house gas emissions, water and energy
consumption, and generation of industrial waste. It is a truly multidisciplinary
frontier area, which closely links the main areas of chemistry and biology.
On the other hand, major challenges still exist when combining these two
principles (biotechnology and green chemistry) still running around the
upstream and downstream processes: from early cell culti-vation, raw
materials supply, to the final challenge to obtain the target material with the
desired characteristics, after separation and pur-ification procedures. All these
steps must be critically evaluated, be-cause they can be intensively
demanding of energy, chemicals and water. Irrespective of whether a
genetically engineered strain is used, both processes must be considered as
they can make a biotechnological process unsustainable, and not green at all
(Chmielowski et al., 2007; Junker et al., 2004).
This work will emphasize some examples of applications of bio-
technology in green chemistry, also discussing some important points related
to its true greenness.
3. Applications
3.1. Biotechnology applied to materials: biopolymers
The petrochemical industry is one of the major sources of air pol-lution
and the increase in GHG emissions (https://www3.epa.gov/cli-
matechange/ghgemissions/). The manufacture of petroleum-based polymers
plays a significant role in generating this pollution (Fu et al., 2003). In
addition, their high durability and strength, in the past seen as “good
qualities”, are now a serious environmental problem, since these polymers
accumulate in the environment, and hundreds of years are required to
completely decompose them, during which they often re-lease toxic
substances that can contaminate soil and groundwater (Webb et al., 2013;
Alay et al., 2016). Biodegradable polymers are a sustainable alternative, since
by definition they are degraded through enzymatic actions that tend to
continuously decrease their molecular masses. The term "degradable" is used
when hydrolysis or enzymatic
M. Fasciotti
degradation occurs, or when the polymer chain cleavage processes occurring
during degradation are not fully known.
Biopolymers are polymer chains synthesized by living organisms and are
therefore perfect examples of the application of biotechnology in green
chemistry, due to several aspects: 1) because they are biode-gradable, they
reduce the accumulation of industrial waste; 2) they tend to generate less
pollutants during production; 3) they can use waste or byproducts as starting
material; 4) they can be less toxic, which in-creases the possibilities of
application in medicine and food, for ex-ample; and 5) they tend to
biodegrade forming innocuous (or less toxic) products to the environment.
Some examples of natural biopolymers are polysaccharides including starch,
chitin/chitosan, cellulose, algi-nate and other carbohydrate polymers. Among
polymers formed from animal proteins are gelatin, wool, silk, collagen and
gluten (Thakur et al., 2014).
The production of biopolymers is an extremely broad subject, especially
those produced by bacteria and those derived from biomass. The use of
bacteria for the production of biopolymers has opened a wide range of
possibilities for the production of different materials, since bacteria are able to
efficiently convert different carbon sources into several types of polymers with
different chemical and mechanical properties. Most bacterial species
synthesize polymers in extracellular form (Rehm and Valla, 1997). Most of
these polymers are formed from inorganic polysaccharides, polyesters,
polyamides and polyanhydrides such as polyphosphates (Pham et al., 2004).
Various biopolymers ob-tained through bacteria are already commercially
produced on a large scale, for example xanthan and dextran polysaccharides.
In 2010, Rehm and co-workers published a review in Nature Reviews
Microbiology on the most important aspects of the production of biopolymers
by bac-teria (Rehm, 2010).
One of the most important applications of biopolymers is in the medical
area. Delplace and Nicolas (2015) reviewed several methods of rendering
vinyl polymers degradable for medical applications. Parti-cularly in medicine,
polymer degradability is extremely important for the choice of materials for a
given application, since biopolymers can be used for the transport of slow
release drugs in the body, as well as for synthetic fabrics and medical
implants. For each application, the bio-polymer must be compatible with the
biological homeostasis of the human body and degrade with a velocity that is
compatible with the function it will perform within the organism. It is difficult
to imagine this level of optimization of these materials without the advent of
biotechnology-related practices and principles.
To illustrate this , a recent paper published in Nature Biotechnology
described a new one-step method for the production of carbohydrate
polylactate-co-glycollate (PLGA) through fermentation by engineered
Escherichia coli, from various stages of genetic modification, to enable
polymerization of D-lactyl-CoA and glycol-CoA in PLGA from xylose and
glucose (Choi et al., 2016).
PLGA is a random copolymer composed of lactic and glycolic acids. It
deserves special attention because it is one of the few commercially available
biopolymers that have been approved by the FDA, being biodegradable,
biocompatible and non-toxic. It is applied primarily in the medical field. The
classic synthesis of PLGA is through a chemical route in which lactic and
glycolic acid dimers are copolymerized in the presence of organometallic
catalysts. The idea of this article was to engineer a strain of Escherichia coli
(E. coli) so that this copolymeriza-tion was performed in only one process,
through fermentative meta-bolism.
To make PLGA directly from carbohydrate fermentation, initially E. coli
was engineered to produce glycolate from glucose by introducing the Dahms
cycle2 into the bacterial metabolism, rather than the native
2 3
The Dahms Cycle is an oxidative route in which D-xylose is oxidized to D-xylyl lactone by
a D-xylose dehydrogenase, followed by a lactonase to hydrolyze the lactone to d-xylic acid.
Then, through the action of xyloneate dehydratase, a water molecule is removed,
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cycle, which involves pentose phosphate, by cloning genes from Cau-lobacter
crescentus. These cells were then engineered to simultaneously use glucose
and xylose to produce the respective lactic and glycolic acids. Third, the
enzyme propionyl-CoA transferase (Pct) was also en-gineered to convert in
vivo the glycolates and lactates to their CoA-derived intermediates. Finally,
another enzyme involved in the synth-esis of polyhydroxyalkanoates (PHA),
an engineered PHA synthase, was used to copolymerize the CoA derivatives
and form the PLGA. The authors then used the same approach, i.e., the novel
metabolic pathway of the engineered E. coli strain resulting from these
various gene in-troduction/exclusion steps, to verify whether it would be
possible to synthesize other copolymers with other monomers having different
compositions. For this, in addition to glycolic acid, they used other chain
acids, such as 2-, 3-, 4-, 5-, and 6-hydroxycarboxylic acids as starting models
.
This work is a very illustrative case study of the massive application of
genetic engineering for the synthesis of biopolymers. For example, to
introduce the Dahms cycle in the metabolism of E. coli, the authors cloned
two genes from another microorganism (C. crescentus), xylB and XylC,
responsible for decoding xylosedehydrogenase and xylonolacto-nase. The E.
coli strain containing this new plasmid3 was able to pro-duce 0.89 g per liter
of glycolic acid from xylose as the only carbon source.
The characteristics of the produced PLGA were also comparable to the
PLGAs chemically synthesized with respect to the monomeric composition
and molecular weight (15,000–25,000 Da), besides having the same thermal
and plastic properties. However, in the chemical route for producing PLGA,
metal catalysts are generally used, and the removal of the byproducts adds
costs to the process. Removal of these catalysts and byproducts is extremely
important for PLGAs used in the medical field, since they will come into
contact with biological fluids and tissues and therefore need to be safe. The
authors stated that this is not as problematic in the metabolic pathway for
PLGA production re-ported in the article. In addition, the approaches used
could be applied to several other biopolymers that can be made from
renewable sources through engineered microorganisms. It is expected that this
area will continue to develop during the next years (Zhu et al., 2016).
Other biopolymers that are receiving considerable attention are those
derived from lignocellulosic biomass, since this material is an extremely
abundant renewable carbon source and does not have a di-rect application
other than burning for the generation of energy. Lignin and cellulose are
generally byproducts generated in industrial processes such as paper, sugar
and ethanol production. They are natural polymers with many possibilities for
conversion into new biopolymers to replace those derived from petroleum
(Fernandes et al., 2013).
The biopolymers that have the greatest potential to replace syn-thetic
products of petrochemical origin are the bioplastics, with appli-cation in the
packaging segment, as they can be recycled as well as being biodegradable
(Soroudi and Jakubowicz, 2013). Polyethylene (PE) made from ethanol from
sugarcane is now being used in several countries, where it is called "I'm
Green" or “Plant bottle”, as a reference to its green origin
(http://www.braskem.com.br/Portal/Principal/
Arquivos/Download/Upload/Catalogo_PE_Verde.pdf). However, in terms of
resource efficiency, ethanol production still waste carbon, since half of carbon
is lost as CO2, making it not ideally sustainable.
The better bioplastics will be those that can be efficiently produced from
biomass and recycled even when mixed with other materials (Hassan et al.,
2013). Without efficient production and recycling bio-technological processes
will not succeed. Moreover, the ethanol
(footnote continued)
resulting in 2-keto-3-deoxyxylonate, which is then cleaved by an aldolase to form pyr-uvate and
glycoaldehyde.
Plasmids are double circular DNA molecules capable of reproducing independently of
chromosomal DNA, used to multiply specific genes. The gene to be replicated is inserted into a
plasmid and then inserted into the microorganism.
M. Fasciotti
produced by fermenting sugarcane still generates residues, mainly ba-gasse,
which in this case can be a good source of carbon for the pro-duction of
biopolymers. Ethanol from sugarcane is already widely used in automotive
fuels (it is the main biofuel used in Brazil). Thus, Shi and Chen published a
paper in Sustainable Chemistry and Engineering (Chen and Shi, 2015),
aiming at the direct transformation of sugarcane ba-gasse into bioplastics by
modifying it through the use of phthalic an-hydride (PA). They performed
homogeneous esterification of cane ba-gasse with phthalic anhydride using 1-
allyl-3-methylthiimidazolium chloride (AMIMCI) – an ionic liquid – as
solvent. Bagasse transforma-tion was obtained in a more chemical than
biological way - which would be done through microorganisms – but it still
poses a problem related to green chemistry, that is, to recycle one of the most
important wastes from a biotechnological process (sugarcane fermentation).
Sugarcane bagasse is a natural composite of lignin, hemicellulose and
cellulose, with several hydroxyls in its structure, which can be chemically
modified to alter its properties. However, the complexity of this biomass
makes research seeking the direct transformation of this material less
common and more challenging. The authors reported that these few studies
were not very successful, because the formed bio-plastics were fragile. The
authors (Chen and Shi, 2015) concluded that the addition of the phthalic
group acts as a plasticizer for sugarcane bagasse, but that improvements still
need to be made to improve the flexibility of this biopolymer without
reducing its tensile strength.
Nevertheless, before planning a new biopolymer, some important aspects
should be taken into account: What plastics really need to be replaced , that is,
what are the principal fossil fuel-based polymers that are causing relevant
damages to the planet? If wanting to replace a non-biodegradable fossil-fuel
polymer, how much of the biobased one is needed to replace the fossil-based
ones? And finally, are sufficient feedstocks (biomass, raw materials, waste and
byproducts) available to produce the biopolymer for replacement? If these
answers tend to be positive, then a greener scenario can be expected (Yu and
Chen, 2008).
3.2. Biofuels
Currently, the energy mix in the world is still based on petroleum
derivatives such as diesel, gasoline and aviation naphtha (IEA, 2007).
However, it is widely argued that the uncertainty of the future avail-ability of
oil, the fluctuations of its value in the international market, and mainly the
pollution generated by burning fossil fuels, requires finding other carbon
sources to replace it. Production of fuels derived from renewable sources of
carbon is one of the main areas of applying biotechnology. Today there is
widespread research to find new re-newable sources and develop new
processes to make various biofuels.
All biofuels have the same conceptual basis: they are produced through
sources that convert solar energy into chemical energy by capturing CO2
through photosynthesis (Sarkar and Shimizu, 2015) to produce a series of
compounds derived from their own metabolic pathways. However, the
sustainability or greenness of a biofuel can be controversial. If one consider
aspects such as how much fertilizer, water and soil will be required to
produce a specific raw material for biofuel; or how efficiently the biomass will
be converted into biofuel; and finally how much greenhouse gases emissions
are produced from seed to fuel, one can conclude that some biofuels do not
satisfy the principles of green chemistry at all (Scharlemann and Laurance,
2008).
Biofuels are currently classified as first, second, and most recently third
and fourth generation (Cherubini, 2010). The first generation is produced
through raw materials that generally compete with the food industry, such as
edible oilseeds, sugarcane, corn, beet and wheat (Gnansounou and Dauriat,
2005). Because of this competition, these biofuels result in both ethical and
political dilemmas, and in addition they still generate waste from their
processing. To overcome these is-sues, the production of second generation
biofuels targets the use of raw materials mainly from agricultural waste or
non-edible biomass sources (Sarkar et al., 2012). However, first-generation
biofuels are the most
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Sustainable Chemistry and Pharmacy 6 (2017) 82–89
widely produced worldwide because of their greater ease of production, such
as biodiesel from soybean, sunflower, corn, canola and re-used vegetable oils,
or animal fats. Biodiesel production in the United States was about 105
million gallons (about 397 million liters) in February 2016
(http://www.eia.gov/biofuels/biodiesel/production/). Bioe-thanol, however, is
mainly obtained from corn, sugarcane, wheat and beet (Nigam and Singh,
2011). The United States and Brazil are the world's largest producers of
bioethanol, which is mostly obtained from sugarcane in Brazil and from corn
in the U.S. In 2015 in Brazil, more than 10 billion liters of sugarcane
bioethanol (http:) was consumed.
Second-generation biofuels generally use lignocellulosic residues from
agriculture or the paper industry, which can be converted into various types of
liquid or gaseous fuels through thermochemical, en-zymatic, pyrolytic and
other processes (Balat et al., 2008; Mood et al., 2013). Examples are the
bioethanol obtained through lignocellulosic biomass and the diesel obtained
by the Fischer-Tropsch process (Stöcker, 2008; Leckel, 2009; van Vliet et al.,
2009).
Third-generation biofuels have recently been developed, involving the use
of algae. Initially, biofuels made from algae were defined as second-
generation, but when it became clear that algae were able to provide much
better yields than processes with other biomasses and through theoretically
simpler production steps, algae were included in a new category. They have
been the subject of massive research for ef-ficient production of biofuels
(Scott et al., 2010). A very interesting advantage of using algae as biomass is
that they can be grown in wastewater such as sewage, acting simultaneously
for bioremediation (Kligerman and Bouwer, 2015).
Fourth-generation fuels, which are the state-of-the-art now, are defined as
photobiological fuels or "electrofuels," and emerge from the incredible
advances in biotechnology and metabolic engineering (Aro, 2016). Both
third- and fourth-generation fuels use photosynthetic mi-croorganisms to
produce fuels: in the third generation, the way to do this is basically to
process the algal biomass to extract the desired biofuel from it, whereas in the
fourth generation metabolic engineering is applied to algae, specially
cyanobacteria and yeast, for the produc-tion of photosynthetic fuels
(Bharathiraja et al., 2015). In practice, the desire is to create a kind of "self-
sustaining biorefinery" in which algae are capable of producing biofuels or
any chemicals only from CO2 and electromagnetic energy from sunlight, a
process also called solar-to-fuel, when the final product can be used as a fuel
directly (Tuller, 2017; Listorti et al., 2009).
Fu and co-workers published a review in 2011 in Energy &
Environmental Science (Lu et al., 2011) summarizing the lit-erature to that
date on the production of biofuels through algae, dis-cussing their genetic
manipulation for metabolic optimization. They discussed various species of
algae from the genomic point of view and described case studies of the
production of biofuels. Currently, there are studies and applications for the
production of: (i) biohydrogen by algae through solar energy and water
(Kruse and Hankamer, 2010); (ii) bioisoprene (2-methyl-1,3-butadiene),
generated from the fixation of CO2 through the mevalonic acid route,
incorporated into bacteria through the cloning of plant genes (Yang et al.,
2012); (iii) bioethanol, mainly by cyanobacteria, but also through metabolic
engineering that integrates genes related, for example, to alcohol
dehydrogenases (ADH) and pyruvate decarboxylases (PDC), allowing yields
comparable to the classic microorganism of alcoholic production, the famous
Sacchar-omyces cerevisiae (John et al., 2011; Kim et al., 2011); (iv) butanol
and other four-carbon alcohols, by Clostridium acetobylicium and Synecho-
coccus elongatus, which after being engineered are used to produce
isobutyraldehyde, isobutanol and butanol directly from CO2 (Astumi et al.,
2008; Atsumi et al., 2009); and (v) biodiesel, the most promising biofuel that
can be produced through algae (Beer et al., 2009; Mata et al., 2010; Chisti,
2008). Some oily algae have 30–60% (w/w) of triacylglycerols on a dry basis
and are synthesized for lipid accumula-tion in their chloroplasts. In order to
obtain better yields in the pro-duction of biodiesel precursor lipids such as
triacylglycerols, the
M. Fasciotti
microalgal species are evaluated in different culture media, containing
different nutrients and physico-chemical conditions such as pH and
temperature, since these parameters have a strong influence on the
composition of the biomass, because algae tend to adapt their meta-bolism to
the growth conditions related to their culture medium.
In a recent study, Nielsen and co-workers (Kerkhoven et al., 2016)
showed that under growth conditions with limited nitrogen supple-mentation,
the regulation of amino acid metabolism controls the flow of lipid
accumulation in the cells of this microorganism, increasing the concentration
of this class of molecules. They studied Yarrowia lipoly-tica, which is an
oleaginous yeast able to accumulate up to 70% of total lipids in its biomass,
with potential for the production of biofuel and other applications in
biotechnology (Gonçalves et al., 2014). Briefly, they cultivated Y. lipolytica
under two conditions: one with restriction of carbon (glucose) and another
with restriction of nitrogen (in the form of ammonia) in the culture medium.
After chemical analyses (by chromatographic and mass spectrometric
techniques) of some classes of lipids, they concluded that the nitrogen
restricted condition favored higher production of lipids, mainly ergosterol,
phosphatidylcholine and triacylglycerols. They then extracted and analyzed
the RNA and verified the changes in gene expression and changes in
transcription that were responsible for the synthesis of lipids under conditions
of nitrogen limitation. This study is important because understanding the
metabolic pathways involved in lipid synthesis, as well as the genes
responsible for these changes in microorganisms, is necessary for the study
with other microorganisms or for cloning for genetic improvement and me-
tabolic optimization.
Also regarding third-generation biofuels, an article published in Nature
Scientific Reports by Slocombe et al. (2015) presented data ob-tained through
a comprehensive and systematic analysis of the biomass composition of
different species of algae. Based on these results, the authors discussed the
best potential biotechnology applications: the production of biofuels, biogas,
bioremediation, food, and so on. To date, this may be the most comprehensive
study of the characterization of algal biomass. The main objective of the work
was to identify the genera, species and strains of algae suitable for large-scale
production in industrial biotechnological processes, such as the production of
biodiesel, biogas, or nutraceutical foods with high nutritional value. The
authors’ goal was to obtain strains capable of growing satisfactorily in salty
water, without the need for additional nutrients such as phos-phates and
nitrates. This would result in a drastic reduction in costs for large-scale
production. For that, they defined a standardized culture medium for the
growth of all strains evaluated, which simulated the physical and chemical
conditions of seawater. Thus, it is important to perform a robust comparative
analysis of the composition of all biomass sources, since the culture medium
has strong influence on the pro-ductivity of certain classes of metabolites, as
previously discussed and exemplified in the work of Nielsen et al. (Chisti,
2008). The authors evaluated 175 algal species, cataloged in CCAP (Culture
Collection of Algae and Protozoa, UK), suitable for long growing times.
Specifically for biodiesel production, the best species/strains were those of
Nannochloropsis oceanica CCAP 849/10 and Chlorella vulgaris CCAP
211/21A. However, the amount of polyunsaturated fatty acids (PUFA) found
in these species/strains can negatively impact the stabi-lity of biodiesel due to
the degree of unsaturation. In general, the de-termined concentrations of
PUFA were higher than those observed for the usual raw materials employed
for biodiesel production, with a mean of 34%, but varying from 4% to 74%
among all species analyzed. This value was 32% in C. vulgaris, but restricted
to three unsaturations per chain at most.
All species of Nanochloropsis were found to have low concentrations of
PUFAs (5–11%) and half was eicosapentaenoic acid (C20:5). Due to the high
concentrations of C16: 0 and C16: 1 acids in N. oceanic species, it was the
one that presented fatty acids with the shortest chains on average among the
analyzed species. This can be seen as an advantage, since acids with long
chains cause cold starting problems in engines due
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Sustainable Chemistry and Pharmacy 6 (2017) 82–89
to the higher melting point of these fatty acid esters. However, C14 fatty
acids, ideal for biodiesel performance, were not found in the species that
produced the highest total fatty acid yield. Some could may serve as models
for genetic engineering, since they presented the highest concentrations of
C14 (but not the highest total fatty acid productivities), such as Prymnesium
parvum, which had 28% C14, and Cylindrotheca sp., which reached up to
30%. In the end of the study, the authors summarized in tables all values
determined for each analyzed parameter and categorized the best species for
each biotechnological application, making the article an important source of
information on the biotechnology of algal biomass.
From the biotechnological point of view, first-generation biofuels use less
sophisticated, or more trivial, biotechnology tools. The pro-duction of
biodiesel through the transesterification of oils is essentially a chemical
process, but when recalling that many oilseeds have un-dergone several stages
of genetic improvement to develop cultivars that have better yield for the
production of biofuels, it is possible to see clearly the role of biotechnology
(Divakara et al., 2010). In this respect, obtaining ethanol from alcoholic
fermentation of sugars is one of the oldest biotechnological processes used. In
fact, second-generation fuels increasingly use more advanced biotechnology
tools, which increase in complexity according to the generation of biofuel
(Keasling and Chou, 2008).
3.3. Organic synthesis
Organic reactions are the center of synthesis and processes of
transforming matter. When conducting a holistic analysis, all processes and
applications already mentioned in this text involve organic reac-tions of
different complexities and for different purposes. Organic synthesis is the
central science behind most of today's industrial pro-cesses and thus is also an
important source of pollutants and toxic wastes. Therefore, the green
chemistry idea calls for changing the way organic synthesis is done today by
developing processes that obey green principles (Bevan and Franssen, 2006).
The ideology of green chemistry generally demands that syntheses be sought
that have greater energy efficiency, selectivity, simplicity, and safety for
nature and human health (Li and Trost, 2008).
The main characteristics of a green organic synthesis are: (i) economy of
atoms, in which the greatest possible amount of reagents must be incorporated
in the product (Trost, 1995); (ii) direct conversion of C–H bonds to C–C
bonds (Trusova et al., 2016); (iii) no use of pro-tective groups, since this
increases the number of steps in the synthesis (Young and Baran, 2009); (iv)
cascade reactions which can incorporate a series of steps for the synthesis of
the product in only one process (Webb and Jamison, 2010); (v) the use of
biocatalysts, which are more selective and efficient, while at the same time
being non-toxic (Reetz, 2013); and (vi) use of less toxic solvents such as
water, supercritical CO2 and ionic liquids (Sheldon, 2005).
In this way, the already defined "white biotechnology" has great
importance within organic synthesis. In many cases, the use of bioca-talysts
as enzymes or microorganisms can be a better alternative route to traditional
synthesis processes, generally allowing greater selectivity and yield (Reetz et
al., 1997) and in most cases being compatible with the use of aqueous media.
In addition, the advances in metagenomics4 have allowed the production of
extremely specific enzymes that cannot be obtained through culturing
microorganisms (Uchiyama and Miyazaki, 2009).
The applications of enzymes and microorganisms as alternative synthetic
routes are a topic apart, due to the numerous applications that
4
Metagenomics is the genomic analysis of the community of microorganisms of a given
environment by independent cultivation techniques. This approach consists of extracting DNA
directly from the environment and constructing a metagenomic library with this mixed genome.
M. Fasciotti
have been developed, mainly for the synthesis of chiral compounds and
amino acids (Nestl et al., 2014). An interesting example that serves as a case
study of organic synthesis performed through microorganisms was published
in 2013 by Jiang and co-workers in Biosource Technology (Jiang et al.,
2013). They proposed the synthesis of succinic acid through Actinobacillus
succinigeses. The biotechnological synthesis of succinic acid has been a
recurrent focus of research, and there are several methods for obtaining it
through microorganisms. These studies were reviewed and published in
Current Opinion in Biotechnology by Lee et al. (Ahn et al., 2016).
The reasons for large number of studies of succinic acid is the fact that it
is an organic molecule with strong industrial interest, since it serves as a
precursor for several other products such as tetrahydrofuran, γ-butyrolactone,
1,4-butanediol, and others (Zeikus et al., 1999). In-dustrially, synthesis of
succinic acid is accomplished by the hydro-genation of maleic acid, oxidation
of 1,4-butanediol alcohol and car-bonylation of ethylene glycol (Cornils and
Lappe, 2005).
The objective of Jiang et al. (Nestl et al., 2014) was to obtain suc-cinic
acid through cellobiose obtained through enzymatic hydrolysis (enzymolysis)
of sugarcane bagasse by the metabolism of Actinobacillus succinogenes,
which is a gram negative bacterium found in bovine rumen and is one of the
most promising bacteria for the production of succinic acid through sugar
sources. Therefore, several production routes through a fermentative process
have been proposed using in-dustrial and agricultural wastes as starting
material for organic synth-esis, since they are renewable sources of carbon
(Bevilaqua et al., 2015). Therefore, this work presented a biotechnological
route to ob-tain succinic acid, using sugars obtained through the hydrolysis of
su-garcane bagasse, making this synthesis renewable and in line with several
concepts of green chemistry.
Another example of alternative synthesis development is the so-called
solar-to-chemical synthesis via microorganisms. In this chemical route, a
microorganism with photosynthetic behavior – native or arti-ficially
introduced to its biological system – is used in the high-effi-ciency synthesis
of several compounds, using CO2 as the carbon source (Woo, 2017).
An interesting example of the implementation of such a system was
published in Science by Yang and co-workers in 2016 (Sakimoto et al., 2016).
The conversion of solar energy into chemicals has a similar scheme to that of
the fourth-generation fuels mentioned in the previous topic. But in this work,
the authors developed a hybrid system that combined the high light capture
efficiency of inorganic semiconductors with the ability of selective and
efficient synthesis of biocatalyst mi-croorganisms. They induced the
photosensitization in an acetogenic non-photosynthetic bacterium, Moorella
thermoacetica, through the biological precipitation of cadmium sulfide
nanoparticles that ag-gregate to the bacterial cells, resulting in the
photosynthetic synthesis of acetic acid from CO2, through the metabolism of
this microorganism. Although it is a trivial molecule, the approach of this
work has great relevance and innovation, since it can be used for the synthesis
of other more complex molecules in future studies.
The authors emphasized that there is a need to improve the capture of
solar energy for sustainable chemical synthesis, and that some major advances
have been made with the development of solid-state inorganic
semiconductors, which generally do this more efficiently than photo-synthetic
processes. However, they do not have the same performance in transforming
this energy into new carbon bonds (mainly in the conversion of CO 2 into
more complex molecules), that is, forming new C–C bonds for organic
synthesis, something that photosynthetic systems do with high efficiency.
Therefore, the approach used in their work aimed to combine the advantages
of these two mechanisms: the ability to capture solar energy of
semiconductors and the high catalytic per-formance of biological systems.
One interesting aspect of this paper is the use of microorganisms to
induce the precipitation of semiconductor nanoparticles. This approach has
some advantages because the semiconductors are, therefore,
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Sustainable Chemistry and Pharmacy 6 (2017) 82–89
biocompatible. It also reduces the costs of their production, since all the
chemical and technological refinement for the synthesis of semi-conductors is
not necessary in this case. Living organisms have the endogenous ability to
perform the synthesis of inorganic materials, such as nanomaterials. One
advantage of this is that they generally can very precisely control the shape
and crystallinity of a developing inorganic material. In this respect, Iverson
and co-workers published the first report of semiconductor nanocrystal
synthesis in bacteria in 2004 (Sweeney et al., 2004). They showed that E. coli,
when incubated with cadmium chloride and sodium sulfide, synthesized
intracellular cad-mium sulfide (CdS) nanocrystals. The CdS nanoparticles
were appar-ently being formed inside bacterial cells following transport of
Cd2+ and S2- ions, and this formation is strongly dependent on the bacterial
growth phase and strain.
The precipitation of semi conductive nanoparticles is mainly per-formed
by photosynthetic microorganisms. However, the authors of the solar-to-
chemical approach (Sakimoto et al., 2016) argued that their metabolic
pathways are sometimes less interesting than the metabolic pathways of non-
photosynthetic microorganisms, because they offer simpler ways of obtaining
a larger diversity of products through the simple reduction of CO2 to new
molecules. In addition, there is a thermodynamic advantage (Fast and
Papoutsakis, 2012) of photo-sensitizing non-photosynthetic bacteria to reduce
CO2 to other organic compounds, caused principally by the metabolic
mechanisms present in these bacteria, such as the called Wood-Ljungdahl
pathway.5 In this mechanism, CO2 is reduced to acetyl-CoA, which is a fairly
common biosynthetic intermediate – and possibly to acetic acid – both of
which can be biotransformed into other more complex organic molecules
through the metabolism of engineered microorganisms.
As previously mentioned, the authors then developed a hybrid system
containing Moorella thermoacetica, a non-photosynthetic bac-teria reducing
CO2, and CdS as biologically precipitated semiconductor nanoparticles. The
photosynthesis of acetic acid by M. thermoacetica and induced by the CdS is
a process in a pot consisting of two stages: first, the precipitation of CdS by
M. thermoacetica is triggered by the addition of Cd2+ ions (by the addition of
Cd(NO3)2 to the culture medium containing glucose during exponential cell
growth), supple-mented with cysteine, which acts as a source of sulfur. M.
thermoacetica uses the electrons generated photochemically by light
irradiation in the CdS to perform the photosynthesis. The absorption of a
photon by the CdS produces a vacancy and a pair of electrons, which are
introduced in the Wood-Ljungdahl cycle to synthesize the acetic acid from
CO2. Cysteine neutralizes the vacancy generated in the semiconductor,
forming its oxidized disulfide, cystine (CySS). All the experimental
procedures - including the quantities of culture media, M. thermoacetica and
Cd2+ ions required to trigger the CdS nanoparticle synthesis, were explained
by the authors in details in the supplementary materials of their manuscript.
These parameters are of great importance and need to be critically evaluated
to consider solar-to-chemical synthesis a sus-tainable process.
Moreover, to further characterize the photosynthetic mechanism induced
in M. thermoacetica, they irradiated the culture by simulating sunlight for 12
hours cycles, as would normally be the case in circadian cycles. They
observed that, curiously, the production of acetic acid did not cease, and
actually increased, even in periods of no light. The ex-planation given by the
authors is possible accumulation of biosynthetic intermediates during periods
of illumination, which are consumed during periods of dark. This shows that
the process could be viable under natural lighting conditions. In addition,
during the natural pho-tosynthesis of photosynthetic organisms, metabolic
alteration occurs in the absence of light, which was not observed in the
photosynthetic
5
In the Wood-Ljungdahl pathway, also known as the acetyl-CoA reduction pathway, some
microorganisms use hydrogen as an electron donor and CO2 as an acceptor for biosynthesis.
M. Fasciotti
mechanism used in this study.
That work showed that the use of hybrid systems of naturally pre-cipitated
semiconductors and photosensitized bacteria can be an effi-cient and self-
sustaining alternative for the synthesis of various che-micals, even using
engineered microorganisms in future studies, for a more diversified
production of other more organic molecules obtained by reducing CO2.
4. Critical view and future perspectives
It is undeniable that biotechnology is a tool for achieving sustain-able
processes and products. This paper discusses general aspects and specific
articles as case studies of the application of biotechnology in "green"
processes to obtain biopolymers and biofuels, and as alternative routes for
organic synthesis of simple molecules.
The work that proposed PLGA synthesis through engineered E. coli (Choi
et al., 2016) is a very representative case of the widespread use of genetic
engineering. To perform the extremely complex metabolic changes employed
in that study, it is certainly necessary to have a highly qualified and
specialized workforce. In addition, the use of ge-netically modified
microorganisms can confront ethical and bureau-cratic conflicts that can slow
down researches advances. The authors emphasizes that this synthetic route
does not use metal catalysts and that this is an advantage primarily for
application in the medical field indeed, but another important aspect to be
discussed is that if the yield is 40%, there is another 60% of bacterial cell
material which needs to be removed. The extraction and purification for the
effective application of this polymeric material should be considered critical
steps for the economic viability of using these biopolymers, mainly for the
medical area.
On the other hand, the process of obtaining a bioplastic from su-garcane
bagasse (reported by Chen and Shi (2015)) is closely aligned with the tenets
of green chemistry, because it produces a bioplastic material from waste
generated on a large scale in the sugar and ethanol industry. It uses an ionic
liquid to dissolve the sugarcane bagasse, since it is practically the only class
of solvents capable of dissolving lignin. Ionic liquids are by definition green
solvents, but this depends greatly on how they are used and can be effectively
reused as solvents, espe-cially in large quantities. A more effective
biotechnological approach to obtaining bioplastics from sugarcane bagasse
would be very interesting, worthy of focus in a future study. For example, one
could prospect for microorganisms that digest the polymer material of the
sugarcane ba-gasse (lignin, cellulose and hemicellulose) and then polymerize
another type of polymer, through genetic engineering and metabolic optimiza-
tion steps, as done for PLGA synthesis.
Regarding the use of algae biomass for the production of third-generation
biofuels, it is undoubtedly promising. However, it is neces-sary to discuss
further the steps of lipid extraction, mainly for the production of biodiesel
(Lemões et al., 2016). For large-scale produc-tion, the strong influence of the
culture medium on the productivity of these algae can be a limiting economic
factor for sale of these biofuels, since the processes in biorefineries can be
very sensitive and unviable due to small changes in biomass growth.
However, research in this area is proceeding so rapidly that it is likely that in
a short time all tech-nological and economic constraints will be overcome.
Undoubtedly, genetic engineering can contribute greatly to the improvement
of lipid production in algae, and even to target the production of the final
biofuels through metabolic routes (Radakovits et al., 2010).
Organic synthesis through fermentative routes is a reality and ex-
pectations are that the use of engineered microorganisms will allow the
synthesis of any organic molecule, from the simplest to the most com-plex,
through renewable carbon sources, or even only atmospheric CO 2. With
respect to the case study of the "solar-to-chemical" synthesis dis-cussed here,
(Sakimoto et al., 2016) the negative aspect is the use of CdS, which although
being an excellent semiconductor due to its high interaction efficiency with
sunlight wavelengths, is extremely toxic to
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Sustainable Chemistry and Pharmacy 6 (2017) 82–89
the environment and human health. Therefore, some improvement by using
more efficient and non-toxic semiconductor nanoparticles needs to be
achieved. But arguably this may be one of the most sustainable synthetic
routes developed.
To sum up, research is being conducted on many fronts and will certainly
lead to important discoveries to mitigate the damage caused by current
unsustainable processes, through the combination of "green" chemistry and
applied biotechnology.
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